Dna polymerase mutants having enhanced template discrimination activity

ABSTRACT

This invention relates to mutant Taq DNA polymerase having an enhanced template discrimination activity compared with an unmodified Taq DNA polymerase of SEQ ID NO.:1, wherein the amino acid sequence of the mutant Taq DNA polymerase consists of substitutions at residue positions 783, 784, or a combination of 783 and 784 of the unmodified Taq DNA polymerase of SEQ ID NO.:1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/386,631, filed Dec. 21, 2016, which is a divisional of U.S. patentapplication Ser. No. 14/542,539, filed Nov. 14, 2014, which claimsbenefit of priority under 35 U.S.C. 119 to U.S. provisional patentapplication Ser. No. 61/904,335, filed Nov. 14, 2013, and entitled “DNAPOLYMERASE MUTANTS HAVING ENHANCED TEMPLATE DISCRIMINATION ACTIVITY,”the contents of which are herein incorporated by reference in itsentirety.

FIELD

This invention relates to mutant DNA polymerases having enhanced primerand/or template discrimination activities and uses of the same forpolymerase-based assays for genetic diagnostic analysis.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. The ASCII copy, created on Sep. 29, 2021, isnamed IDT01-003-US-DIV2_ST25.txt, and is 362,522 bytes in size.

BACKGROUND

The ability to accurately diagnose a given genetic condition and topredictably treat a genetically-based disorder requires reliable methodsfor accurately determining genetic sequence information. Manygenetically-based disorders are associated with single nucleotidepolymorphisms (SNP's) in protein coding genes. The presence of SNP'sassociated with a genetically-based disorder, such as a cancer, can bedifficult to detect owing to the small numbers of genetically alteredcells in the population that encode the allele(s) (“rare alleles”).

Polymerase-based assays, such as the polymerase chain reaction (PCR),have important impact and widespread use in genetic diagnostics andmolecular medicine. Polymerases synthesize DNA sequences by the additionof nucleotides to the 3′ end of a short oligonucleotide (abbreviated to“primer” in the following). The primer is hybridized to the singlestranded sequence that is going to be amplified (“template”). DNApolymerases catalyze formation of a phosphodiester bond between the3′-oxygen at the 3′-terminus of the primer and the incomingdeoxynucleoside triphosphate (“dNTP”). This chemical reaction (“primerextension”) adds a nucleotide to the primer (e.g., to the nascentgrowing DNA chain). Primer extension is base specific, in that thedeoxynucleoside triphosphate that is complementary to the base in thetemplate is added to the primer. The fidelity of DNA polymerase enzymesis very high and the rate of mutations introduced into the replicatedDNA strand is low; however, the precise error rate varies betweendifferent DNA polymerase enzymes and these rates have been wellcharacterized. The extension reaction can be repeated until the end ofthe template is reached.

The majority of polymerase-based assays for detecting SNP's rely uponhaving the polymerase enzyme discriminate between at least two differentsubstrates. The first substrate contains the desired SNP that is to bedetected; the second substrate contains the normal nucleotide that isnot to be detected. Polymerase-based discrimination can be achieved byproviding the first substrate as the preferred polymerase-competentsubstrate for assay. This discrimination can be maximized to the extentthat the first substrate is the only polymerase-competent substratepresent for assay.

Many strategies are known in the art for establishing conditions thatfavor polymerase-based discrimination among substrates having minimalnucleotide differences, such as those containing only single nucleotidedifferences. One strategy relies on a polymerase's inability toefficiently initiate synthesis on substrates lacking a 3′-pairednucleotide on the primer. An allele-specific primer design is a primerin which the 3′-nucleotide forms a perfect match with the complementarybase at the location containing the SNP-containing allele and forms amismatched pairing when annealed to an allele lacking the SNP. Theprimer:template for the SNP-containing allele serves as the preferredpolymerase-competent substrate since the polymerase can efficientlyinitiate primed synthesis from such substrates. Examples of thesestrategies are described in Chen et al., “Single nucleotide polymorphismgenotyping: biochemistry, protocol, cost and throughput”Pharmacogenomics J. 3(2):77-96 (2003); Kwok et al., “Detection of singlenucleotide polymorphisms” Curr. Issues Mol. Biol. 5(2):43-60 (April2003); Shi, “Technologies for individual genotyping: detection ofgenetic polymorphisms in drug targets and disease genes” Am. J.Pharmacogenomics 2(3):197-205 (2002); and Kwok, “Methods for genotypingsingle nucleotide polymorphisms” Annu. Rev. Genomics Hum. Genet.2:235-58 (2001). A strategy to improve selectivity for this class ofallele-specific PCR primers is to introduce a second mutation at thepenultimate base, next to the 3′-terminal nucleotide of the primer(i.e., next to the SNP site). As before, the 3′-terminal residue willeither be a match or mismatch to the base under interrogation in thesample nucleic acid (SNP), but now the primer will either have twoadjacent mismatches to the target (both 3′-terminal and penultimatebase) or a single mismatch to the target (at only the penultimate base,with the 3′-terminal base being a match). See, for example, Newton etal., “Analysis of any point mutation in DNA. The amplificationrefractory mutation systems (ARMS)” Nucleic Acids Res. 17(7):2503-15(1989). Yet another strategy to improve selectivity for this class ofallele-specific PCR primers is to employ a chemically modified nucleicacid residue at the 3′-end of the primer, such as a locked nucleic acid(LNA), which reduces the ability of DNA polymerase to initiate DNAsynthesis from a 3′-terminal mismatch. See, for example, Latorra et al.,“SNP genotyping using 3′ locked nucleic acid (LNA) primers” Human Mut.22(1):79-85 (2003).

Template substrate discrimination can be enhanced in polymerase-basedassays by requiring a second nucleic acid enzyme catalyze formation ofone or more primers for use in the polymerase-based assay. In one suchassay, the ligase chain reaction assay, a DNA ligase is used with apolymerase to detect a template containing a SNP. Since polymerase-basedassays require primers having a minimum length to hybridize to thetemplate substrate, a DNA ligase can be used to generate polymeraseprimers from sub-optimal length oligonucleotides. The assay relies uponhybridizing two probes directly over the SNP polymorphic site, wherebyligation can occur if the probes are identical to the target DNA. Twoprobes are designed; an allele-specific probe which hybridizes to thetarget DNA so that its 3′ base is situated directly over the SNPnucleotide and a second probe that hybridizes the template downstream inthe complementary strand of the SNP polymorphic site providing a 5′ endfor the ligation reaction. If the allele-specific probe matches thetarget DNA, it will fully hybridize to the target DNA and ligation canoccur. Ligation does not generally occur in the presence of a mismatched3′-base. Once the oligonucleotide product is formed, it can serve as aprimer or as a template for polymerase-based assays. Examples of thisstrategy are described in Barany F. “Genetic disease detection and DNAamplification using cloned thermostable ligase.” Proc Natl Acad Sci USA.1991 Jan. 1; 88(1):189-93 and Wiedmann M., Wilson W. J., Czajka J., LuoJ., Barany F., Batt C. A. “Ligase chain reaction (LCR)—overview andapplications.” PCR Methods and Applications 1994 Feb; 3(4):S51-64.

Since a polymerase-competent substrate requires a primer:template havingan available 3′-hydroxyl group on the primer, another strategy known inthe art, RNase H-based PCR (rhPCR), can be used for improvingpolymerase-based discrimination. The rhPCR method makes use of RNase Henzymes to convert a primer lacking a 3′-hydroxyl group (“blockedprimer”) or a primer that is otherwise disabled and cannot support PCRto a primer containing a 3′-hydroxyl group (“unblocked primer”) that cansupport PCR. A blocked primer in rhPCR includes an oligonucleotidehaving a blocked 3′-end or other modification which prevents eitherpriming or template function of the oligonucleotide and an internal RNAresidue, which serves as a cleavage site. Type II RNase H recognizesannealed primer:template duplexes containing these blocked primers andcleaves the primer strand 5′ of the RNA residue to generate a3′-hydroxyl group at the adjacent DNA residue. Since RNase H enzymes donot cleave substrates containing an unpaired RNA reside at a mismatchedsite, allele-specific template discrimination can be achieved byplacement of the RNA residue at the location complementary to the SNP onthe selected allele template. The resultant Type II RNase H cleavageproduct can serve as a polymerase competent substrate. Examples of thisenzyme cleaving strategy, similar RNase H strategies, and methods ofblocking primer extension or inhibiting template function and therebydisabling PCR are described in U.S. Pat. No. 7,112,406 to Behlke et al.,entitled POLYNOMIAL AMPLIFICATION OF NUCLEIC ACIDS, U.S. Pat. No.5,763,181 to Han et al., entitled CONTINOUS FLUOROMETRIC ASSAY FORDETECTING NUCLEIC ACID CLEAVAGE, U.S. Pat. No. 7,135,291 to Sagawa etal., entitled METHOD OF DETECTING NUCLEOTIDE POLYMORPHISM; U.S. Pat.App. No. 20090068643 to Behlke and Walder, entitled DUAL FUNCTIONPRIMERS FOR AMPLIFYING DNA AND METHODS OF USE; and U.S. Pat. App. No.20100167353 to Walder et al., entitled RNASE H-BASED ASSAYS UTILIZINGMODIFIED RNA MONOMERS.

The field has focused on substrate-based approaches, such as thoseexemplified above, for improving detection of genetic differences andrare alleles. Yet the sensitivity of polymerase-based assays remainslimited by the formation of non-specific amplification products thatarise from ectopic or aberrant primer-related extension productsindependent of the desired templates. The inherent reactivity of thepolymerase appears largely responsible for producing such artifactsduring amplification.

Any further improvements in mismatch discrimination may thereforerequire a modified polymerase enzyme endowed with inherently better3′-nucleotide discrimination when used with one or more of the describedstrategies. A modified polymerase enzyme having activity differing fromthe unmodified form can be prepared by chemical or enzymaticmodification of the protein or mutagenesis of corresponding gene thatencodes the protein. The latter approach is generally preferred owing tothe fact that genetically altered genes encoding a given mutant proteincan be stably maintained, expressed and purified to yield enzymepreparations having well-characterized properties.

While an unbiased mutagenesis strategy can be used to generate a libraryof mutant polymerase genes, this approach has certain disadvantages.Many millions of mutant enzymes must be screened for activity andsuccess is often dependent upon the chance that effective mutations arepresent in the limited pool generated by random mutagenesis. Directgenetic selection methods are not sufficiently sensitive for identifyingmutations that pertain to secondary functions falling outside of anessential polymerase activity. The vast majority of mutant polymerasegenes in a positive selection assay will likely encode protein thatretains the functional attributes of the normal polymerase enzyme. Thus,secondary screening procedures that use biochemical assays must be doneto identify whether any mutant polymerases have the desired activity.Notwithstanding the technical difficulties of setting up the initialselection process, the attendant costs associated with performing thesecondary screens using biochemical assays is prohibitive if more thanone hundred clones need to be purified and assayed.

An alternative approach is to apply a biased mutagenesis strategy thatis specifically targeted to a preselected region of a gene implicated infunction. In this approach, one first identifies genetic regions by aselection method. One such selection method is a comparativephylogenetic analysis of a particular gene that is required fororganisms of diverse origins. The principle of comparative phylogeneticanalysis is premised on the hypothesis that diverse organisms will notshare protein coding sequences in essential genes unless those sequencesare evolutionary constrained for reasons related to essential proteinfunction.

A phylogenetic comparative analysis of genes encoding DNA polymerasescan provide insights about possible amino acid residues important topolymerase functions. The overall folding pattern of DNA polymerasesresembles the human right hand and contains three distinct subdomains ofpalm, fingers, and thumb. (See, for example. Beese et al., Science260:352-355, 1993; Patel et al., Biochemistry 34:5351-5363, 1995). Whilethe structure of the fingers and thumb subdomains vary greatly betweenpolymerases that differ in size and in cellular functions, the catalyticpalm subdomains are all superimposable. For example, motif A, whichinteracts with the incoming dNTP and stabilizes the transition stateduring chemical catalysis, is superimposable with a root mean deviationof about one Ångström among mammalian pol α and prokaryotic pol I familyDNA polymerases (Wang et al., Cell 89:1087-1099, 1997). Motif A beginsstructurally at an antiparallel β-strand containing predominantlyhydrophobic residues and continues to an α-helix. The primary amino acidsequence of DNA polymerase active sites is exceptionally conserved.

In addition to being well-conserved, the active site of DNA polymeraseshas also been shown to be relatively mutable, capable of accommodatingcertain amino acid substitutions without reducing DNA polymeraseactivity significantly. (See, e.g., U.S. Pat. No. 6,602,695). Suchmutant DNA polymerases can offer various selective advantages in, e.g.,diagnostic and research applications comprising nucleic acid synthesisreactions. We identify mutations in protein sequence using thesingle-letter amino acid codes and an integer number that indicateslocation of the mutation from the beginning of the protein sequence. Thesingle-letter amino acids codes are well known in the art, e.g., Stryeret al., Biochemistry, 5.sup.th ed., Freeman and Company (2002). As anexample, aspartic acid (“D”) is changed to glycine (“G”) in D580G mutantand the change is located 580 amino acids from the beginning of theprotein sequence.

Reichert et al. conducted a comparative phylogenetic analysis ofthermoactive DNA polymerases from thermophilic bacteria, wherein theprotein coding sequences of DNA Polymerase I enzymes were aligned forthirteen phylogenetically distinct species. The analysis revealed thateight amino acid positions within a 15-amino acid long motif located atamino acid positions 645-685 (in reference to Thermus sp. Z05 DNApolymerase (“Z05 DNA Polymerase”) might tolerate alterations withoutcompromising core enzyme function.

Comparative phylogenetic analysis does not provide specific functionalinformation pertaining non-conserved amino acids, other than to suggestthat non-conserved amino acids are not likely critical to core enzymefunctions. For that reason, specific mutations were introduced into arecombinant gene encoding a variant of the Z05 DNA Polymerase (“Z05D580G polymerase”) and the resultant Z05 D580G polymerase mutants werescreened for their ability to provide a reduced ability to extend anoligonucleotide primer with a 3′-mismatch to a template. Reichert et al.found that one such mutant, Z05 D580G V667E polymerase, displayed betterdiscrimination (˜2-fold) than the parental Z05 D580G polymerase. SeeU.S. Pat. App. No. 2012/0015405 to Reichert et al., entitled DNAPOLYMERASES WITH INCREASED 3′-MISMATCH DISCRIMINATION.

The comparative phylogenetic analysis has limitations with respect toidentifying DNA polymerase activities that display improved3′-nucleotide discrimination. This is due to the fact that all DNApolymerases of a given enzyme class are confronted with similar templatesubstrates and nucleotide pools across the spectrum of phylogeneticallydiverse organisms. Given the fact that all DNA polymerases must display3′-nucleotide mismatch discrimination to preserve high fidelityreplication of daughter template strands, it is not surprising that onecan apply comparative phylogenetic analysis to identify possible aminoacid positions that might affect mismatch discrimination. For templatesubstrates having different 3′-end modifications that are presented to apolymerase only in a biochemical assay, such as those used in severalPCR-based assays for rare allele detection, there is a need foridentifying DNA polymerases having improved 3′-nucleotidediscrimination.

Taq DNA Polymerase is an enzyme that was discovered in Thermus aquaticusbacterium (Chien, A., et al., J Bacteriol. 1976, 127: 1550-1557). Theenzyme is classified as deoxyribonucleic acid polymerase, class I(enzyme code, EC 2.7.7.7). Its catalytic activity is to amplify DNAsequences. The peptide and gene sequences of Taq DNA polymerase enzymeisolated from nature are well known in prior art and are listed in Table1 (Lawyer, F. C., et al., J. Biol. Chem. 1989, 264: 6427-6437; Genbankdatabase ID J04639.1).

TABLE 1 DNA and amino acid sequence of native Taq DNA polymerase. TypeSequence ProteinMRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKED sequenceGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGY (N to CEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGL terminus)RPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKI[SEQ ID NO: 1]LAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE DNA sequenceaagctcagat ctacctgcct gagggcgtcc ggttccagct ggcccttccc (5′ to 3′)gagggggaga gggaggcgtt tctaaaagcc cttcaggacg ctacccgggg [SEQ ID NO: 2]gcgggtggtg gaagggtaac atgaggggga tgctgcccct ctttgagcccaagggccggg tcctcctggt ggacggccac cacctggcct accgcaccttccacgccctg aagggcctca ccaccagccg gggggagccg gtgcaggcggtctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggacgcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggcctacgggggg tacaaggcgg gccgggcccc cacgccggag gactttccccggcaactcgc cctcatcaag gagctggtgg acctcctggg gctggcgcgcctcgaggtcc cgggctacga ggcggacgac gtcctggcca gcctggccaagaaggcggaa aaggagggct acgaggtccg catcctcacc gccgacaaagacctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgaggggtacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccgaccagtgggcc gactaccggg ccctgaccgg ggacgagtcc gacaaccttcccggggtcaa gggcatcggg gagaagacgg cgaggaagct tctggaggagtgggggagcc tggaagccct cctcaagaac ctggaccggc tgaagcccgccatccgggag aagatcctgg cccacatgga cgatctgaag ctctcctgggacctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaaaggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttgagtttggcagc ctcctccacg agttcggcct tctggaaagc cccaaggccctggaggaggc cccctggccc ccgccggaag gggccttcgt gggctttgtgctttcccgca aggagcccat gtgggccgat cttctggccc tggccgccgccagggggggc cgggtccacc gggcccccga gccttataaa gccctcagggacctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggccctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgcctacctcctg gacccttcca acaccacccc cgagggggtg gcccggcgctacggcgggga gtggacggag gaggcggggg agcgggccgc cctttccgagaggctcttcg ccaacctgtg ggggaggctt gagggggagg agaggctcctttggctttac cgggaggtgg agaggcccct ttccgctgtc ctggcccacatggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtccctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcctggccggccac cccttcaacc tcaactcccg ggaccagctg gaaagggtcctctttgacga gctagggctt cccgccatcg gcaagacgga gaagaccggcaagcgctcca ccagcgccgc cgtcctggag gccctccgcg aggcccaccccatcgtggag aagatcctgc agtaccggga gctcaccaag ctgaagagcacctacattga ccccttgccg gacctcatcc accccaggac gggccgcctccacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctccgatcccaac ctccagaaca tccccgtccg caccccgctt gggcagaggatccgccgggc cttcatcgcc gaggaggggt ggctattggt ggccctggactatagccaga tagagctcag ggtgctggcc cacctctccg gcgacgagaacctgatccgg gtcttccagg aggggcggga catccacacg gagaccgccagctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgggcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccgcctctcccag gagctagcca tcccttacga ggaggcccag gccttcattgagcgctactt tcagagcttc cccaaggtgc gggcctggat tgagaagaccctggaggagg gcaggaggcg ggggtacgtg gagaccctct tcggccgccgccgctacgtg ccagacctag aggcccgggt gaagagcgtg cgggaggcggccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctcatgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggccaggatgctc cttcaggtcc acgacgagct ggtcctcgag gccccaaaagagagggcgga ggccgtggcc cggctggcca aggaggtcat ggagggggtgtatcccctgg ccgtgcccct ggaggtggag gtggggatag gggaggactggctctccgcc aaggagtgat accacc

Taq DNA polymerase extends primers composed from deoxyribonucleotides,however, some chemical modifications of the primer are tolerated and donot decrease much the efficiency of primer extension reactions. Forexample, when the nucleotide at 3′ primer terminus is ribonucleotideinstead of deoxyribonucleotide, Taq DNA polymerase can extend suchprimer with significant efficiency and speed.

BRIEF SUMMARY OF THE INVENTION

In one aspect, a mutant Taq DNA polymerase having an enhanced templatediscrimination activity compared with an unmodified Taq DNA polymeraseis provided. The amino acid sequence of the mutant Taq DNA polymeraseincludes at least one substitution at residue positions 783 or 784 ofthe unmodified Taq DNA polymerase.

In another aspect, a mutant thermostable DNA polymerase having anenhanced template discrimination activity compared with an unmodifiedthermostable DNA polymerase is provided. The amino acid sequence of themutant thermostable DNA polymerase includes at least one substitution atresidue positions orthologous to positions 783 or 784 of the unmodifiedTaq DNA polymerase.

In another aspect, a mutant DNA polymerase having an enhanced templatediscrimination activity compared with the corresponding unmodified DNApolymerase is provided, wherein the mutant DNA polymerase includes athermostable polymerase. The amino acid sequence of the mutant DNApolymerase peptide includes at least one substitution at residuepositions orthologous to positions 783 or 784 of the unmodified Taq DNApolymerase, wherein the mutant DNA polymerase is selected from the groupof species consisting of E. coli, Eubacterium siraeum, Clostridiumleptum, Enterococcus, Facklamia hominis, Bacillus anthracis and Bacilluscereus ATCC 10987.

In another aspect, a mutant non-VH-related DNA polymerase having anenhanced template discrimination activity compared with its unmodifiednon-VH-related DNA polymerase counterpart is provided, wherein themutant non-VH-related DNA polymerase includes a thermostable polymerase.The amino acid sequence of the mutant non-VH-related DNA polymeraseincludes at least one substitution at residue positions orthologous toreside positions 783 and/or 784 of the unmodified Taq DNA polymerase.

In another aspect, recombinant nucleic acid encoding any of the mutantDNA polymerases of described above is provided.

In another aspect, a method for conducting primer extension is provided.The method includes the step of contacting a mutant DNA polymerase asdescribed above with a primer, a polynucleotide template, and nucleosidetriphosphates under conditions suitable for a primer extension method,thereby producing an extended primer.

In another aspect, a kit for producing an extended primer, comprising:at least one container providing a mutant DNA polymerase as describedabove.

In another aspect, a reaction mixture is provided that includes a mutantDNA polymerase as described above, at least one primer, a polynucleotidetemplate, and nucleoside triphosphates.

In another aspect, a method for performing rhPCR is provided thatincludes the step of performing primer extension with a mutant DNApolymerase as described above.

In another aspect, a mutant Taq DNA polymerase having an enhancedtemplate discrimination activity compared with an unmodified Taq DNApolymerase is provided. The amino acid sequence of the mutant Taq DNApolymerase comprises one of following selected substitutions: (1) A661E;I665W; F667L [SEQ ID NO:87]; (2) V783F [SEQ ID NO:83]; (3) H784Q [SEQ IDNO:85]; (4) V783L; H784Q [SEQ ID NO:89]; (5) H784A [SEQ ID NO:147]; (6)H784S [SEQ ID NO:149]; (7) H784I [SEQ ID NO:155]; (8) H784T [SEQ IDNO:151], (9) H784V [SEQ ID NO:153]; (10) H784M [SEQ ID NO:157]; (11)H784F [SEQ ID NO:159]; or (12) H784Y [SEQ ID NO:161].

In another aspect, a mutant Taq DNA polymerase having a deleted 5′exonuclease domain (KlenTaq) and containing additional mutations, havingan enhanced template discrimination activity compared with an unmodifiedTaq DNA polymerase is provided. The amino acid sequence of the mutantTaq DNA polymerase comprises one of following selected substitutions:(1) A661E; 1665W; F667L [SEQ ID NO: 170]; (2) V783F [SEQ ID NO: 172];(3) H784Q [SEQ ID NO: 174]; (4) V783L; H784Q [SEQ ID NO: 176]; (5) H784S[SEQ ID NO: 178]; or (6) H784Y [SEQ ID NO: 180].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a model of the active site constructed from the known TaqDNA polymerase PDB ID 2KTQ crystal structure. The polymerase backbone isdisplayed using ribbons. The “C2′” label indicates the 2′ carbon atom atthe primer terminal nucleotide. The dNTP is binding in the pocket abovethe primer.

FIG. 2A shows gel images depicting purified protein for the Taq DNApolymerase mutants and wild type Taq DNA polymerase. Legend: Aliquots ofpurified recombinant proteins were separated by polyacrylamide gelelectrophoresis (PAGE) and stained with Coomassie Brilliant Blue. TheMarker lane (M) is indicated, showing protein size markers identified inkilodaltons (kDa).

FIG. 2B shows gel images depicting purified protein for the Taq DNApolymerase mutants and wild type Taq DNA polymerase. Legend as in FIG.2A.

FIG. 2C shows gel images depicting purified protein for the Taq DNApolymerase mutants and wild type Taq DNA polymerase. Legend as in FIG.2A.

FIG. 2D shows gel images depicting purified protein for the Taq DNApolymerase mutants and wild type Taq DNA polymerase. Legend as in FIG.2A.

FIG. 3A shows graphical representations of the allele-specific PCR(AS-PCR) data from Tables 10 and 31 for wild type OptiTaq (sub-panel(i)), Mutant ID 2 (sub-panel (ii)) and Mutant ID 3 (sub-panel (iii)).Legend: Average ΔCq values obtained from AS-PCR reactions plus/minusstandard deviation (error bars) are shown (ΔCq=Cq mismatch−Cq match)comparing mismatch discrimination of the wild-type OptiTaq with themutant Taq DNA polymerases. All possible pairwise mismatch basecombinations are included. The base identity of the SNP site in thetarget nucleic acid is indicated on the X-axis (A, G, C, T) along withthe 3′-DNA residue of the AS-PCR reverse primer employed (dA, dG, dC,dT).

FIG. 3B shows graphical representations of the allele-specific PCR(AS-PCR) data from Tables 10 and 31 for wild type OptiTaq (sub-panel(i)), Mutant ID 10 (sub-panel (ii)) and Mutant ID 18 (sub-panel (iii)).Legend as in FIG. 3A.

FIG. 3C shows graphical representations of the allele-specific PCR(AS-PCR) data from Tables 10 and 31 for wild type OptiTaq (sub-panel(i)), Mutant ID 3 (sub-panel (ii)) and Mutant ID 20 (sub-panel (iii)).Legend as in FIG. 3A.

FIG. 3D shows graphical representations of the allele-specific PCR(AS-PCR) data from Tables 10 and 31 for wild type OptiTaq (sub-panel(i)), Mutant ID21 (sub-panel (ii)) and Mutant ID 22 (sub-panel (iii)).Legend as in FIG. 3A.

FIG. 3E. shows graphical representations of the allele-specific PCR(AS-PCR) data from Tables 10 and 31 for wild type OptiTaq (sub-panel(i)), Mutant ID 24 (sub-panel (ii)) and Mutant ID 26 (sub-panel (iii)).Legend as in FIG. 3A.

FIG. 3F shows graphical representations of the allele-specific PCR(AS-PCR) data from Tables 10 and 31 for wild type OptiTaq (sub-panel(i)), Mutant ID 27 (sub-panel (ii)) and Mutant ID 29 (sub-panel (iii)).Legend as in FIG. 3A.

FIG. 3G shows graphical representations of the allele-specific PCR(AS-PCR) data from Tables 10 and 31 for wild type OptiTaq (sub-panel(i)) and Mutant ID 30 (sub-panel (ii)). Legend as in FIG. 3A.

FIG. 4 shows a graphical representation of the ΔCq values (Table 13)obtained from comparing qPCR results using primers ending with a 3′-RNAresidue and primers ending with a 3′-DNA residue for wild type OptiTaqwith four mutant Taq DNA polymerases, where ΔCq=Cq 3′-RNA-Cq 3′-DNA; rAis compared with dA, rC is compared with dC, rG is compared with dG, andrU is compared with dT. Identity of each DNA polymerase studied is shownon the X-axis.

FIG. 5A shows graphical representations of the ΔCq values (Tables 20 and34) obtained from comparing mismatch discrimination between wild typeOptiTaq with Mutant ID 2, Mutant ID 3, Mutant ID 10, and Mutant ID 18Taq DNA polymerases detecting a human genomic DNA SNP in the SMAD7 gene(NM_005904, C/T SNP, rs4939827) using Gen1 RDDDDx blocked-cleavableprimers in a quantitative rhPCR assay. ΔCq=[Cq mismatch−Cq match].Legend: Identity of each DNA polymerase studied is shown on the X-axis.The RDDDDx blocked-cleavable primer contained either a rC or rU residueas the cleavable base, specific for the “C” or “T” allele, as indicated.

FIG. 5B shows graphical representations of the ΔCq values (Tables 20 and34) obtained from comparing mismatch discrimination between wild typeOptiTaq with Mutant ID 3, Mutant ID 20, Mutant ID 21, Mutant ID 22, andMutant ID 24 Taq DNA polymerases detecting a human genomic DNA SNP inthe SMAD7 gene (NM_005904, C/T SNP, rs4939827) using Gen1 RDDDDxblocked-cleavable primers in a quantitative rhPCR assay. ΔCq=[Cqmismatch−Cq match]. Legend as in FIG. 5A.

FIG. 5C shows graphical representations of the ΔCq values (Tables 20 and34) obtained from comparing mismatch discrimination between wild typeOptiTaq with Mutant ID 3, Mutant ID 26, Mutant ID 27, Mutant ID 29, andMutant ID 30 Taq DNA polymerases detecting a human genomic DNA SNP inthe SMAD7 gene (NM_005904, C/T SNP, rs4939827) using Gen1 RDDDDxblocked-cleavable primers in a quantitative rhPCR assay. ΔCq=[Cqmismatch−Cq match]. Legend as in FIG. 5A.

FIG. 6A shows a graphical representation of the ΔCq values (Tables 21and 35) obtained from comparing mismatch discrimination between wildtype OptiTaq with Mutant ID 2, Mutant ID 3, Mutant ID 10, and Mutant ID18 Taq DNA polymerases detecting a human genomic DNA SNP in the SMAD7gene (NM_005904, C/T SNP, rs4939827) using Gen2 RDxxD blocked-cleavableprimers in a quantitative rhPCR assay. ΔCq=[Cq mismatch−Cq match].Legend: Identity of each DNA polymerase studied is shown on the X-axis.The RDxxD blocked-cleavable primer contained either a rC or rU residueas the cleavable base, specific for the “C” or “T” allele, as indicated.

FIG. 6B shows a graphical representation of the ΔCq values (Tables 21and 35) obtained from comparing mismatch discrimination between wildtype OptiTaq with Mutant ID 3, Mutant ID 20, Mutant ID 21, Mutant ID 22,and Mutant ID 24 Taq DNA polymerases detecting a human genomic DNA SNPin the SMAD7 gene (NM_005904, C/T SNP, rs4939827) using Gen2 RDxxDblocked-cleavable primers in a quantitative rhPCR assay. ΔCq=[Cqmismatch−Cq match]. Legend as in FIG. 6A.

FIG. 6C shows a graphical representation of the ΔCq values (Tables 21and 35) obtained from comparing mismatch discrimination between wildtype OptiTaq with Mutant ID 3, Mutant ID 26, Mutant ID 27, Mutant ID 29,and Mutant ID 30 Taq DNA polymerases detecting a human genomic DNA SNPin the SMAD7 gene (NM_005904, C/T SNP, rs4939827) using Gen2 RDxxDblocked-cleavable primers in a quantitative rhPCR assay. ΔCq=[Cqmismatch−Cq match]. Legend as in FIG. 6A.

FIG. 7A shows graphical representations of the allele-specific PCR(AS-PCR) data from Tables 10 and 31 for OptiTaq KlenTaq (Mutant ID 37)(sub-panel (i)), Mutant ID 38 (sub-panel (ii)) and Mutant ID 39(sub-panel (iii)). Legend: Average ΔCq values obtained from AS-PCRreactions plus/minus standard deviation (error bars) are shown (ΔCq=Cqmismatch−Cq match) comparing mismatch discrimination of the wild-typeOptiTaq with four mutant Taq DNA polymerases. All possible pairwisemismatch base combinations are included. The base identity of the SNPsite in the target nucleic acid is indicated on the X-axis (A, G, C, T)along with the 3′-DNA residue of the AS-PCR reverse primer employed (dA,dG, dC, dT).

FIG. 7B. shows graphical representations of the allele-specific PCR(AS-PCR) data from Tables 10 and 31 for OptiTaq KlenTaq (Mutant ID 37)(sub-panel (i)), Mutant ID 40 (sub-panel (ii)) and Mutant ID 41(sub-panel (iii)). Legend as in FIG. 7A.

FIG. 7C. shows graphical representations of the allele-specific PCR(AS-PCR) data from Tables 10 and 31 for OptiTaq KlenTaq (Mutant ID 37)(sub-panel (i)), Mutant ID 42 (sub-panel (ii)) and Mutant ID 43(sub-panel (iii)). Legend as in FIG. 7A.

DETAILED DESCRIPTION

The current invention provides novel thermostable DNA polymerases,including specific examples derived from Thermus aquaticus (Taq) DNApolymerase. These polymerases offer improvements to existing methods fornucleic acid amplification, genotyping, and detection of rare alleles.New assay formats comprising the use of these novel thermostable DNApolymerases are also provided.

Definitions

To aid in understanding the invention, several terms are defined below.

Terms used herein are intended as “open” terms (for example, the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.).

The articles “a” and “an” refer to one or to more than one (for example,to at least one) of the grammatical object of the article.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Exemplary degrees of error are within 20-25 percent(%), typically, within 10%, and more typically, within 5% of a givenvalue or range of values.

Furthermore, in those instances where a convention analogous to “atleast one of A,B and C, etc.” is used, in general such a construction isintended in the sense of one having ordinary skill in the art wouldunderstand the convention (for example, “a system having at least one ofA, B and C” would include but not be limited to systems that have Aalone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together.). It will be further understoodby those within the art that virtually any disjunctive word and/orphrase presenting two or more alternative terms, whether in thedescription or figures, should be understood to contemplate thepossibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or ‘B or “A and B.”

All language such as “from,” “to,” “up to,” “at least,” “greater than,”“less than,” and the like, include the number recited and refer toranges which can subsequently be broken down into sub-ranges.

A range includes each individual member. Thus, for example, a grouphaving 1-3 members refers to groups having 1, 2, or 3 members.Similarly, a group having 6 members refers to groups having 1, 2, 3, 4,or 6 members, and so forth.

The modal verb “may” refers to the preferred use or selection of one ormore options or choices among the several described embodiments orfeatures contained within the same. Where no options or choices aredisclosed regarding a particular embodiment or feature contained in thesame, the modal verb “may” refers to an affirmative act regarding how tomake or use and aspect of a described embodiment or feature contained inthe same, or a definitive decision to use a specific skill regarding adescribed embodiment or feature contained in the same. In this lattercontext, the modal verb “may” has the same meaning and connotation asthe auxiliary verb “can.”

The term “conventional” or “natural” when referring to nucleic acidbases, nucleoside triphosphates, or nucleotides refers to those whichoccur naturally in the polynucleotide being described (i.e., for DNAthese are dATP, dGTP, dCTP and dTTP). Additionally, dITP, and7-deaza-dGTP are frequently utilized in place of dGTP and 7-deaza-dATPcan be utilized in place of dATP in in vitro DNA synthesis reactions,such as sequencing. Collectively, these may be referred to as dNTPs.

The term “unconventional” or “modified” when referring to a nucleic acidbase, nucleoside, or nucleotide includes modification, derivations, oranalogues of conventional bases, nucleosides, or nucleotides thatnaturally occur in a particular polynucleotide. Certain unconventionalnucleotides are modified at the 2′ position of the ribose sugar incomparison to conventional dNTPs. Thus, although for RNA the naturallyoccurring nucleotides are ribonucleotides (i.e., ATP, GTP, CTP, UTP,collectively rNTPs), because these nucleotides have a hydroxyl group atthe 2′ position of the sugar, which, by comparison is absent in dNTPs,as used herein, ribonucleotides are unconventional nucleotides assubstrates for DNA polymerases. As used herein, unconventionalnucleotides include, but are not limited to, compounds used asterminators for nucleic acid sequencing. Exemplary terminator compoundsinclude but are not limited to those compounds that have a 2′,3′ dideoxystructure and are referred to as dideoxynucleoside triphosphates. Thedideoxynucleoside triphosphates ddATP, ddTTP, ddCTP and ddGTP arereferred to collectively as ddNTPs.

The term “nucleotide,” in addition to referring to the naturallyoccurring ribonucleotide or deoxyribonucleotide monomers, shall hereinbe understood to refer to related structural variants thereof, includingderivatives and analogs, that are functionally equivalent with respectto the particular context in which the nucleotide is being used (e.g.,hybridization to a complementary base), unless the context clearlyindicates otherwise.

The terms “nucleic acid” and “oligonucleotide,” as used herein, refer topolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and to any other type ofpolynucleotide which is an N glycoside of a purine or pyrimidine base.There is no intended distinction in length between the terms “nucleicacid”, “oligonucleotide” and “polynucleotide”, and these terms will beused interchangeably. These terms refer only to the primary structure ofthe molecule. Thus, these terms include double- and single-stranded DNA,as well as double- and single-stranded RNA. For use in the presentinvention, an oligonucleotide also can comprise nucleotide analogs inwhich the base, sugar or phosphate backbone is modified as well asnon-purine or non-pyrimidine nucleotide analogs.

Oligonucleotides can be prepared by any suitable method, includingdirect chemical synthesis by a method such as the phosphotriester methodof Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiestermethod of Brown et al., 1979, Meth. Enzymol. 68:109-151; thediethylphosphoramidite method of Beaucage et al., 1981, TetrahedronLett. 22:1859-1862; and the solid support method of U.S. Pat. No.4,458,066, each incorporated herein by reference. A review of synthesismethods of conjugates of oligonucleotides and modified nucleotides isprovided in Goodchild, 1990, Bioconjugate Chemistry 1(3): 165-187,incorporated herein by reference.

The term “primer,” as used herein, refers to an oligonucleotide capableof acting as a point of initiation of DNA synthesis under suitableconditions. Such conditions include those in which synthesis of a primerextension product complementary to a nucleic acid strand is induced inthe presence of four different nucleoside triphosphates and an agent forextension (e.g., a DNA polymerase or reverse transcriptase) in anappropriate buffer and at a suitable temperature. Primer extension canalso be carried out in the absence of one or more of the nucleosidetriphosphates in which case an extension product of limited length isproduced. As used herein, the term “primer” is intended to encompass theoligonucleotides used in ligation-mediated reactions, in which oneoligonucleotide is “extended” by ligation to a second oligonucleotidewhich hybridizes at an adjacent position. Thus, the term “primerextension”, as used herein, refers to both the polymerization ofindividual nucleoside triphosphates using the primer as a point ofinitiation of DNA synthesis and to the ligation of two oligonucleotidesto form an extended product.

A primer is preferably a single-stranded DNA. The appropriate length ofa primer depends on the intended use of the primer but typically rangesfrom 6 to 50 nucleotides, preferably from 15-35 nucleotides. Shortprimer molecules generally require cooler temperatures to formsufficiently stable hybrid complexes with the template. A primer neednot reflect the exact sequence of the template nucleic acid, but must besufficiently complementary to hybridize with the template. The design ofsuitable primers for the amplification of a given target sequence iswell known in the art and described in the literature cited herein.

Primers can incorporate additional features which allow for thedetection or immobilization of the primer but do not alter the basicproperty of the primer, that of acting as a point of initiation of DNAsynthesis. For example, primers may contain an additional nucleic acidsequence at the 5′ end which does not hybridize to the target nucleicacid, but which facilitates cloning or detection of the amplifiedproduct. The region of the primer which is sufficiently complementary tothe template to hybridize is referred to herein as the hybridizingregion. Primers may incorporate modified residues other than DNA, solong as these alternations do not impede priming or templatefunctionality.

The phrase “3′-nucleotide discrimination” refers to a property of a DNApolymerase to catalyze a primer extension reaction with greaterspecificity for deoxyribonucleotides and less efficiently when thenucleotide at the primer 3′ terminus was chemically modified. Forexample, a mutated Taq DNA polymerase that displays 3′-nucleotidediscrimination exhibits selectivity for deoxyribonucleotide primer andsuppressed catalytic activity when the primer is for example modifiedwith ribonucleotides.

The term “3′-mismatch discrimination” refers to a property of a DNApolymerase to distinguish a fully complementary sequence from amismatch-containing (nearly complementary) sequence where the nucleicacid to be extended (for example, a primer or other oligonucleotide) hasa mismatch at the 3′ terminus of the nucleic acid compared to thetemplate to which the nucleic acid hybridizes. In some embodiments, thenucleic acid to be extended comprises a mismatch at the 3′ end relativeto the fully complementary sequence.

The term “rare allele discrimination” refers to a property of a DNApolymerase to preferentially replicate a first nucleic acid in apopulation of nucleic acids that includes a plurality of a secondnucleic acid, wherein the first nucleic acid is under-represented in thepopulation of nucleic acids relative to the plurality of a secondnucleic acid. Typically, the first nucleic acid may be under-representedin the population of nucleic acids that contain a plurality of a secondnucleic acids by a ratio of the first nucleic acid to the second nucleicacid in the range from about 1:10 to about 1:1,000,000, including 1:100,1:1,000; 1:10,000 and 1:100,000, among other ratios. Typically, thoughnot exclusively, a polymerase having rare allele discrimination can beused to detect a SNP difference between a first nucleic acid and asecond nucleic acid, as further elaborated herein.

The phrase “template discrimination activity” refers to a DNA polymerasehaving at least one of 3′-nucleotide discrimination, 3′-mismatchdiscrimination, rare allele discrimination and combinations thereof.

The phrase “enhanced template discrimination activity” refers to a DNApolymerase having at least one of 3′-nucleotide discrimination,3′-mismatch discrimination and rare allele discrimination, orcombinations thereof, wherein the DNA polymerase displays greateractivity than a reference DNA polymerase. For example, a DNA polymerasemutant having “enhanced template discrimination activity” displays atleast one of 3′-nucleotide discrimination, 3′-mismatch discrimination,rare allele discrimination and combinations thereof that is greater thanthe corresponding activity of the naturally-occurring, wild-type DNApolymerase from which the DNA polymerase mutant was derived.

A “template discrimination activity assay” refers to an assay forassessing the ability of a polymerase to discriminate between twotemplates that differ in one or more variables. Assays designed toreveal 3′-nucleotide discrimination, 3′-mismatch discrimination or rareallele discrimination are examples of template discrimination activityassays.

The term “quantification cycle value,” denoted as Cq, refers to theamplification cycle number at which positive signal is first detected.

The term “discrimination quantification cycle value,” denoted as ΔCq,refers to a calculated difference between a first reference state and asecond reference state, wherein both the first and second referencestates differ in terms of only one variable. For examples, a first andsecond reference states can refer to identical polymerase reactions thatdiffer in polymerases, such as a wild-type polymerase and a polymerasemutant, that differ in primer template nucleotide sequence, such as amismatched primer template and a matched primer template, or that differin primer template 3′-nucleotide ribose structure, such as a primertemplate containing a 3′-deoxyribose moiety and a primer templatecontaining a 3′-ribose moiety.

The term “differential discrimination quantification cycle value,”denoted as ΔΔCq, refers to a calculated difference between a firstdiscrimination quantification cycle value and a second discriminationquantification cycle value for polymerase reactions that differ in twovariables. In the context of the present disclosure, the ΔΔCq value is ameasure of the improvement that a given polymerase mutant displaysrelative to the wild-type polymerase in a template discriminationactivity assay. A preferred ΔΔCq value depends upon the nature of theassay, but generally a preferred ΔΔCq value is at least 1.0 and istypically greater than 1.0.

The terms “target, “target sequence”, “target region”, and “targetnucleic acid,” as used herein, are synonymous and refer to a region orsequence of a nucleic acid which is to be amplified, sequenced ordetected.

The term “template” refers to a nucleic acid that includes at least onesingle stranded region. The term “template” as it modifies “substrate”refers to a nucleic acid that is used in a hybridization reaction toanneal with a primer and/or an extension reaction with a polymerase.

The term “hybridization,” as used herein, refers to the formation of aduplex structure by two single-stranded nucleic acids due tocomplementary base pairing. Hybridization can occur between fullycomplementary nucleic acid strands or between “substantiallycomplementary” nucleic acid strands that contain minor regions ofmismatch. Conditions under which hybridization of fully complementarynucleic acid strands is strongly preferred are referred to as “stringenthybridization conditions” or “sequence-specific hybridizationconditions”. Stable duplexes of substantially complementary sequencescan be achieved under less stringent hybridization conditions; thedegree of mismatch tolerated can be controlled by suitable adjustment ofthe hybridization conditions. Those skilled in the art of nucleic acidtechnology can determine duplex stability empirically considering anumber of variables including, for example, the length and base paircomposition of the oligonucleotides, ionic strength, and incidence ofmismatched base pairs, following the guidance provided by the art (see,e.g., Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; Wetmur, 1991,Critical Review in Biochem. and Mol. Biol. 26(3/4):227-259; and Owczarzyet al., 2008, Biochemistry, 47: 5336-5353, which are incorporated hereinby reference).

The term “amplification reaction” refers to any chemical reaction,including an enzymatic reaction, which results in increased copies of atemplate nucleic acid sequence or results in transcription of a templatenucleic acid. Amplification reactions include reverse transcription, thepolymerase chain reaction (PCR), including Real Time PCR (see U.S. Pat.Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)), and the ligase chain reaction(LCR) (see Barany et al., U.S. Pat. No. 5,494,810). Exemplary“amplification reactions conditions” or “amplification conditions”typically comprise either two or three step cycles. Two step cycles havea high temperature denaturation step followed by ahybridization/elongation (or ligation) step. Three step cycles comprisea denaturation step followed by a hybridization step followed by aseparate elongation or ligation step.

An “amino acid” refers to any monomer unit that can be incorporated intoa peptide, polypeptide, or protein. As used herein, the term “aminoacid” includes the following twenty natural or genetically encodedalpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine(Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine(Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (Hisor H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K),methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P),serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine(Tyr or Y), and valine (Val or V). In cases where “X” residues areundefined, these should be defined as “any amino acid.” The structuresof these twenty natural amino acids are shown in, e.g., Stryer et al.,Biochemistry, 5.sup.th ed., Freeman and Company (2002), which isincorporated by reference. Additional amino acids, such asselenocysteine and pyrrolysine, can also be genetically coded for(Stadtman (1996) “Selenocysteine,” Annu Rev Biochem. 65:83-100 and Ibbaet al. (2002) “Genetic code: introducing pyrrolysine,” Curr Biol.12(13):R464-R466, which are both incorporated by reference). The term“amino acid” also includes unnatural amino acids, modified amino acids(e.g., having modified side chains and/or backbones), and amino acidanalogs. See, e.g., Zhang et al. (2004) “Selective incorporation of5-hydroxytryptophan into proteins in mammalian cells,” Proc. Natl. Acad.Sci. U.S.A. 101(24):8882-8887, Anderson et al. (2004) “An expandedgenetic code with a functional quadruplet codon” Proc. Natl. Acad. Sci.U.S.A. 101(20):7566-7571, Ikeda et al. (2003) “Synthesis of a novelhistidine analogue and its efficient incorporation into a protein invivo,” Protein Eng. Des. Sel. 16(9):699-706, Chin et al. (2003) “AnExpanded Eukaryotic Genetic Code,” Science 301(5635):964-967, James etal. (2001) “Kinetic characterization of ribonuclease S mutantscontaining photoisomerizable phenylazophenylalanine residues,” ProteinEng. Des. Sel. 14(12):983-991, Kohrer et al. (2001) “Import of amber andochre suppressor tRNAs into mammalian cells: A general approach tosite-specific insertion of amino acid analogues into proteins,” Proc.Natl. Acad. Sci. U.S.A. 98(25):14310-14315, Bacher et al. (2001)“Selection and Characterization of Escherichia coli Variants Capable ofGrowth on an Otherwise Toxic Tryptophan Analogue,” J. Bacteriol.183(18):5414-5425, Hamano-Takaku et al. (2000) “A Mutant Escherichiacoli Tyrosyl-tRNA Synthetase Utilizes the Unnatural Amino AcidAzatyrosine More Efficiently than Tyrosine,” J. Biol. Chem.275(51):40324-40328, and Budisa et al. (2001) “Proteins with{beta}-(thienopyrrolyl)alanines as alternative chromophores andpharmaceutically active amino acids,” Protein Sci. 10(7):1281-1292,which are each incorporated by reference.

The term “residue” is synonymous and interchangeable with “amino acid”or “nucleotide” depending upon context.

As used herein, a “polymerase” refers to an enzyme that catalyzes thepolymerization of nucleotides. Generally, the enzyme will initiatesynthesis at the 3′-end of the primer annealed to a nucleic acidtemplate sequence. “DNA polymerase” catalyzes the polymerization ofdeoxyribonucleotides. Known DNA polymerases include, for example,Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene,108:1), E. coli DNA polymerase I (Lecomte and Doubleday, 1983, NucleicAcids Res. 11:7505), T7 DNA polymerase (Nordstrom et al., 1981, J. Biol.Chem. 256:3112), Thermus thermophilus (Tth) DNA polymerase (Myers andGelfand 1991, Biochemistry 30:7661), Bacillus stearothermophilus DNApolymerase (Stenesh and McGowan, 1977, Biochim Biophys Acta 475:32),Thermococcus litoralis (Tli) DNA polymerase (also referred to as VentDNA polymerase, Cariello et al., 1991, Nucleic Acids Res, 19: 4193),Thermotoga maritima (Tma) DNA polymerase (Diaz and Sabino, 1998 Braz J.Med. Res, 31:1239), Thermus aquaticus (Taq) DNA polymerase (Chien etal., 1976, J. Bacteoriol, 127: 1550), Pyrococcus kodakaraensis KOD DNApolymerase (Takagi et al., 1997, Appl. Environ. Microbiol. 63:4504),JDF-3 DNA polymerase (Patent application WO 0132887), and PyrococcusGB-D (PGB-D) DNA polymerase (Juncosa-Ginesta et al., 1994,Biotechniques, 16:820). The polymerase activity of any of the aboveenzymes can be determined by means well known in the art.

The term “thermostable polymerase,” refers to an enzyme that is stableto heat, is heat resistant, and retains sufficient activity to effectsubsequent polynucleotide extension reactions and does not becomeirreversibly denatured (inactivated) when subjected to the elevatedtemperatures for the time necessary to effect denaturation ofdouble-stranded nucleic acids. The heating conditions necessary fornucleic acid denaturation are well known in the art and are exemplifiedin, e.g., U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,965,188, which areincorporated herein by reference. As used herein, a thermostablepolymerase is suitable for use in a temperature cycling reaction such asthe polymerase chain reaction (“PCR”). Irreversible denaturation forpurposes herein refers to permanent and complete loss of enzymaticactivity. For a thermostable polymerase, enzymatic activity refers tothe catalysis of the combination of the nucleotides in the proper mannerto form polynucleotide extension products that are complementary to atemplate nucleic acid strand. Thermostable DNA polymerases fromthermophilic bacteria include, e.g., DNA polymerases from Thermusaquaticus, among others.

The term “thermoactive” refers to an enzyme that maintains catalyticproperties at temperatures commonly used for reverse transcription oranneal/extension steps in RT-PCR and/or PCR reactions (i.e., 45-80° C.).Thermostable enzymes are those which are not irreversibly inactivated ordenatured when subjected to elevated temperatures necessary for nucleicacid denaturation. Thermoactive enzymes may or may not be thermostable.Thermoactive DNA polymerases can be DNA or RNA dependent fromthermophilic species or from mesophilic species including, but notlimited to, Escherichia coli, Moloney murine leukemia viruses, and Avianmyoblastosis virus.

As used herein, a primer is “specific,” for a target sequence if, whenused in an amplification reaction under sufficiently stringentconditions, the primer hybridizes primarily to the target nucleic acid.Typically, a primer is specific for a target sequence if theprimer-target duplex stability is greater than the stability of a duplexformed between the primer and any other sequence found in the sample.One of skill in the art will recognize that various factors, such assalt conditions as well as base composition of the primer and thelocation of the mismatches, will affect the specificity of the primer,and that routine experimental confirmation of the primer specificitywill be needed in many cases. Hybridization conditions can be chosenunder which the primer can form stable duplexes only with a targetsequence. Thus, the use of target-specific primers under suitablystringent amplification conditions enables the selective amplificationof those target sequences which contain the target primer binding sites.

The term “non-specific amplification,” as used herein, refers to theamplification of nucleic acid sequences other than the target sequencewhich results from primers hybridizing to sequences other than thetarget sequence and then serving as a substrate for primer extension.The hybridization of a primer to a non-target sequence is referred to as“non-specific hybridization” and is apt to occur especially during thelower temperature, reduced stringency, pre-amplification conditions, orin situations where there is a variant allele in the sample having avery closely related sequence to the true target as in the case of asingle nucleotide polymorphism (SNP).

The term “primer dimer,” as used herein, refers to atemplate-independent non-specific amplification product, which isbelieved to result from primer extensions wherein another primer servesas a template. Although primer dimers frequently appear to be aconcatamer of two primers, i.e., a dimer, concatamers of more than twoprimers also occur. The term “primer dimer” is used herein genericallyto encompass a template-independent non-specific amplification product.

The term “reaction mixture,” as used herein, refers to a solutioncontaining reagents necessary to carry out a given reaction. An“amplification reaction mixture”, which refers to a solution containingreagents necessary to carry out an amplification reaction, typicallycontains oligonucleotide primers and a DNA polymerase or ligase in asuitable buffer. A “PCR reaction mixture” typically containsoligonucleotide primers, a DNA polymerase (most typically a thermostableDNA polymerase), dNTPs, and a divalent metal cation in a suitablebuffer. A reaction mixture is referred to as complete if it contains allreagents necessary to enable the reaction, and incomplete if it containsonly a subset of the necessary reagents. It will be understood by one ofskill in the art that reaction components are routinely stored asseparate solutions, each containing a subset of the total components,for reasons of convenience, storage stability, or to allow forapplication-dependent adjustment of the component concentrations, andthat reaction components are combined prior to the reaction to create acomplete reaction mixture. Furthermore, it will be understood by one ofskill in the art that reaction components are packaged separately forcommercialization and that useful commercial kits may contain any subsetof the reaction components which includes the blocked primers of theinvention.

The terms “non-activated” or “inactivated,” as used herein, refer to aprimer or other oligonucleotide that is incapable of participating in aprimer extension reaction or a ligation reaction because either DNApolymerase or DNA ligase cannot interact with the oligonucleotide fortheir intended purposes. In some embodiments when the oligonucleotide isa primer, the non-activated state occurs because the primer is blockedat or near the 3′-end so as to prevent primer extension. When specificgroups are bound at or near the 3′-end of the primer, DNA polymerasecannot bind to the primer and extension cannot occur. A non-activatedprimer is, however, capable of hybridizing to a substantiallycomplementary nucleotide sequence.

The term “activated,” as used herein, refers to a primer or otheroligonucleotide that is capable of participating in a reaction with DNApolymerase or DNA ligase. A primer or other oligonucleotide becomesactivated after it hybridizes to a substantially complementary nucleicacid sequence and is cleaved to generate a functional 3′- or 5′-end sothat it can interact with a DNA polymerase or a DNA ligase. For example,when the oligonucleotide is a primer, and the primer is hybridized to atemplate, a 3′-blocking group can be removed from the primer by, forexample, a cleaving enzyme such that DNA polymerase can bind to the 3′end of the primer and promote primer extension.

The term “cleavage domain” or “cleaving domain,” as used herein, aresynonymous and refer to a region located between the 5′ and 3′ end of aprimer or other oligonucleotide that is recognized by a cleavagecompound, for example a cleavage enzyme, that will cleave the primer orother oligonucleotide. For the purposes of this invention, the cleavagedomain is designed such that the primer or other oligonucleotide iscleaved only when it is hybridized to a complementary nucleic acidsequence, but will not be cleaved when it is single-stranded. Thecleavage domain or sequences flanking it may include a moiety that a)prevents or inhibits the extension or ligation of a primer or otheroligonucleotide by a polymerase or a ligase, b) enhances discriminationto detect variant alleles, or c) suppresses undesired cleavagereactions. One or more such moieties may be included in the cleavagedomain or the sequences flanking it.

The term “RNase H cleavage domain,” as used herein, is a type ofcleavage domain that contains one or more ribonucleic acid residue or analternative analog which provides a substrate for an RNase H. An RNase Hcleavage domain can be located anywhere within a primer oroligonucleotide, and is preferably located at or near the 3′-end or the5′-end of the molecule.

An “RNase H1 cleavage domain” generally contains at least threeconsecutive RNA residues. An “RNase H2 cleavage domain” may contain oneRNA residue, a sequence of contiguously linked RNA residues or RNAresidues separated by DNA residues or other chemical groups. Forexample, an RNase H2 cleavage domain may include a 2′-fluoronucleosideresidue, among others.

The terms “cleavage compound,” or “cleaving agent” as used herein,refers to any compound that can recognize a cleavage domain within aprimer or other oligonucleotide, and selectively cleave theoligonucleotide based on the presence of the cleavage domain. Thecleavage compounds utilized in the invention selectively cleave theprimer or other oligonucleotide comprising the cleavage domain only whenit is hybridized to a substantially complementary nucleic acid sequence,but will not cleave the primer or other oligonucleotide when it issingle stranded. The cleavage compound cleaves the primer or otheroligonucleotide within or adjacent to the cleavage domain. The term“adjacent,” as used herein, means that the cleavage compound cleaves theprimer or other oligonucleotide at either the 5′-end or the 3′ end ofthe cleavage domain. Cleavage reactions preferred in the invention yielda 5′-phosphate group and a 3′-OH group.

In a preferred embodiment, the cleavage compound is a “cleaving enzyme.”A cleaving enzyme is a protein or a ribozyme that is capable ofrecognizing the cleaving domain when a primer or other nucleotide ishybridized to a substantially complementary nucleic acid sequence, butthat will not cleave the complementary nucleic acid sequence (i.e., itprovides a single strand break in the duplex). The cleaving enzyme willalso not cleave the primer or other oligonucleotide comprising thecleavage domain when it is single stranded. Examples of cleaving enzymesare RNase H enzymes and other nicking enzymes.

The term “nicking,” as used herein, refers to the cleavage of only onestrand of the double-stranded portion of a fully or partiallydouble-stranded nucleic acid. The position where the nucleic acid isnicked is referred to as the “nicking site” (NS). A “nicking agent” (NA)is an agent that nicks a partially or fully double-stranded nucleicacid. It may be an enzyme or any other chemical compound or composition.In certain embodiments, a nicking agent may recognize a particularnucleotide sequence of a fully or partially double-stranded nucleic acidand cleave only one strand of the fully or partially double-strandednucleic acid at a specific position (i.e., the NS) relative to thelocation of the recognition sequence. Such nicking agents (referred toas “sequence specific nicking agents”) include, but are not limited to,nicking endonucleases (e.g., Nt.BstNBI).

A “nicking endonuclease” (NE), as used herein, thus refers to anendonuclease that recognizes a nucleotide sequence of a completely orpartially double-stranded nucleic acid molecule and cleaves only onestrand of the nucleic acid molecule at a specific location relative tothe recognition sequence. In such a case the entire sequence from therecognition site to the point of cleavage constitutes the “cleavagedomain”.

The term “blocking group,” as used herein, refers to a chemical moietythat is bound to the primer or other oligonucleotide such that anamplification reaction does not occur. For example, primer extensionand/or DNA ligation does not occur. Once the blocking group is removedfrom the primer or other oligonucleotide, the oligonucleotide is capableof participating in the assay for which it was designed (PCR, ligation,sequencing, etc.). Thus, the “blocking group” can be any chemical moietythat inhibits recognition by a polymerase or DNA ligase. The blockinggroup may be incorporated into the cleavage domain but is generallylocated on either the 5′- or 3′-side of the cleavage domain. Theblocking group can be comprised of more than one chemical moiety. In thepresent invention the “blocking group” is typically removed afterhybridization of the oligonucleotide to its target sequence.

The term “blocked-cleavable primer” refers to a primer that is inactiveor inactivated for priming DNA synthesis from a polymerase owing to thepresence of a blocking group at or near the 3′-terminus of the primer. Ablocked-cleavable primer can be converted into a competent primer byremoving the blocking group at or near the 3′-terminus of the primer bya cleavage compound or a cleaving agent (for example, a cleaving enzyme)resulting in an active or activated primer.

An RDDDDx blocked-cleavable primer (also known as “generation 1” or “Gen1” blocked-cleavable primer) refers to a blocked-cleavable primer havingat its 3′-terminus the sequence RDDDDx, wherein R is an RNA base, D is aDNA base and x is a C3 spacer group.

An RDxxD blocked-cleavable primer (also known as “generation 2” or “Gen2” blocked-cleavable primer) refers to a blocked-cleavable primer havingat its 3′-terminus the sequence RDxxD, wherein R is an RNA base, D is aDNA base and x is a C3 spacer group.

The term “fluorescent generation probe” refers either to a) anoligonucleotide having an attached fluorophore and quencher, andoptionally a minor groove binder or to b) a DNA binding reagent such asSYBR™ Green dye.

The terms “fluorescent label” or “fluorophore” refers to compounds witha fluorescent emission maximum between about 350 and 900 nm. A widevariety of fluorophores can be used, including but not limited to: 5-FAM(also called 5-carboxyfluorescein; also calledSpiro(isobenzofuran-1(3H), 9′-(9H)xanthene)-5-carboxylicacid,3′,6′-dihydroxy-3-oxo-6-carboxyfluorescein);5-Hexachloro-Fluorescein;([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloyl-fluoresceinyl)-6-carboxyli-cacid]); 6-Hexachloro-Fluorescein;([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloylfluoresceinyl)-5-carboxylicacid]); 5-Tetrachloro-Fluorescein;([4,7,2′,7′-tetra-chloro-(3′,6′-dipivaloylfluoresceinyl)-5-carboxylicacid]); 6-Tetrachloro-Fluorescein;([4,7,2′,7′-tetrachloro-(3′,6′-dipivaloylfluoresceinyl)-6-carboxylicacid]); 5-TAMRA (5-carboxytetramethylrhodamine); Xanthylium,9-(2,4-dicarboxyphenyl)-3,6-bis(dimethyl-amino); 6-TAMRA(6-carboxytetramethylrhodamine);9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid); 1,5-IAEDANS(5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid); Cy5(Indodicarbocyanine-5); Cy3 (Indo-dicarbocyanine-3); and BODIPY FL(2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-proprionicacid); Quasar®-670 dye (Biosearch Technologies); Cal Fluor® Orange dye(Biosearch Technologies); Rox dyes; Max dyes (Integrated DNATechnologies), as well as suitable derivatives thereof.

As used herein, the term “quencher” refers to a molecule or part of acompound, which is capable of reducing the emission from a fluorescentdonor when attached to or in proximity to the donor. Quenching may occurby any of several mechanisms including fluorescence resonance energytransfer, photo-induced electron transfer, paramagnetic enhancement ofintersystem crossing, Dexter exchange coupling, and exciton couplingsuch as the formation of dark complexes. Fluorescence is “quenched” whenthe fluorescence emitted by the fluorophore is reduced as compared withthe fluorescence in the absence of the quencher by at least 10%, forexample, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,99.9% or more. A number of commercially available quenchers are known inthe art, and include but are not limited to DABCYL, Black Hole™Quenchers (BHQ-1, BHQ-2, and BHQ-3), Iowa Black® FQ and Iowa Black® RQ.These are so-called dark quenchers. They have no intrinsic fluorescencein the wavelength range from 300 to 900 nm, virtually eliminatingbackground problems seen with other quenchers such as TAMRA which isintrinsically fluorescent.

The term “ligation” as used herein refers to the covalent joining of twopolynucleotide ends. In various embodiments, ligation involves thecovalent joining of a 3′ end of a first polynucleotide (the acceptor) toa 5′ end of a second polynucleotide (the donor). Ligation results in aphosphodiester bond being formed between the polynucleotide ends. Invarious embodiments, ligation may be mediated by any enzyme, chemical,or process that results in a covalent joining of the polynucleotideends. In certain embodiments, ligation is mediated by a ligase enzyme.

As used herein, “ligase” refers to an enzyme that is capable ofcovalently linking the 3′-hydroxyl group of one polynucleotide to the 5′phosphate group of a second polynucleotide. Examples of ligases includeE. coli DNA ligase, T4 DNA ligase, etc.

The ligation reaction can be employed in DNA amplification methods suchas the “ligase chain reaction” (LCR), also referred to as the “ligaseamplification reaction” (LAR), see Barany, Proc. Natl. Acad. Sci.,88:189 (1991); and Wu and Wallace, Genomics 4:560 (1989) incorporatedherein by reference. In LCR, four oligonucleotides, two adjacentoligonucleotides which uniquely hybridize to one strand of the targetDNA, and a complementary set of adjacent oligonucleotides, thathybridize to the opposite strand are mixed and DNA ligase is added tothe mixture. In the presence of the target sequence, DNA ligase willcovalently link each set of hybridized molecules. Importantly, in LCR,two oligonucleotides are ligated together only when they base-pair withsequences without gaps. Repeated cycles of denaturation, hybridizationand ligation amplify a short segment of DNA. A mismatch at the junctionbetween adjacent oligonucleotides inhibits ligation. As in otheroligonucleotide ligation assays this property allows LCR to be used todistinguish between variant alleles such as SNPs. LCR has also been usedin combination with PCR to achieve enhanced detection of single-basechanges, see Segev, PCT Public. No. WO9001069 (1990).

The term “unmodified form,” in the context of the Taq DNA polymerase, isa term used herein for purposes of defining a host cell-specific,codon-optimized Taq DNA polymerase gene that expresses Taq DNApolymerase in the host cell. The term “unmodified form” refers to afunctional DNA polymerase that has the amino acid sequence of thenaturally occurring polymerase. The term “unmodified form” includes afunctional DNA polymerase in a recombinant form.

The term “mutant”, in the context of DNA polymerases disclosed, means apolypeptide, typically recombinant, that comprises one or more aminoacid substitutions relative to a corresponding, naturally-occurring formor unmodified form of DNA polymerase.

“Recombinant”, as used herein, refers to an amino acid sequence or anucleotide sequence that has been intentionally modified by recombinantmethods. By the term “recombinant nucleic acid” herein is meant anucleic acid, originally formed in vitro, in general, by themanipulation of a nucleic acid by endonucleases, in a form not normallyfound in nature. Thus an isolated, mutant DNA polymerase nucleic acid,in a linear form, or an expression vector formed in vitro by ligatingDNA molecules that are not normally joined, are both consideredrecombinant for the purposes of this invention. It is understood thatonce a recombinant nucleic acid is made and reintroduced into a hostcell, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. A “recombinant protein”is a protein made using recombinant techniques, i.e., through theexpression of a recombinant nucleic acid as depicted above.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation.

The term “vector” refers to a piece of DNA, typically double-stranded,which may have inserted into it a piece of foreign DNA. The vector maybe, for example, of plasmid origin. Vectors contain “replicon”polynucleotide sequences that facilitate the autonomous replication ofthe vector in a host cell. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell, which, for example,replicates the vector molecule, encodes a selectable or screenablemarker, or encodes a transgene. The vector is used to transport theforeign or heterologous DNA into a suitable host cell. Once in the hostcell, the vector can replicate independently of or coincidental with thehost chromosomal DNA, and several copies of the vector and its insertedDNA can be generated. In addition, the vector can also contain thenecessary elements that permit transcription of the inserted DNA into anmRNA molecule or otherwise cause replication of the inserted DNA intomultiple copies of RNA. Some expression vectors additionally containsequence elements adjacent to the inserted DNA that increase thehalf-life of the expressed mRNA and/or allow translation of the mRNAinto a protein molecule. Many molecules of mRNA and polypeptide encodedby the inserted DNA can thus be rapidly synthesized.

The term “affinity tag” refers to a short polypeptide sequence thatpermits detection and/or selection of the polypeptide sequence. For thepurposes of this disclosure, a recombinant gene that encodes arecombinant DNA polymerase may include an affinity tag. In particular,the affinity tag is positioned typically at either the N-terminus orC-terminus of the coding sequence for a DNA polymerase through the useof recombination technology. Exemplary affinity tags include polyhistine(for example,(His6),), glutathione-S-transferase (GST), HaloTag®,AviTag, Calmodulin-tag, polyglutamate tag, FLAG-tag, HA-tag, Myc-tag,S-tag, SBP-tag, Softag 3, V5 tag, Xpress tag, among others.

The term “host cell” refers to both single-cellular prokaryote andeukaryote organisms (e.g., bacteria, yeast, and actinomycetes) andsingle cells from higher order plants or animals when being grown incell culture. Exemplary suitable host cells include E. coli, S.cerevisiae and S. frugiperda.

As used herein, “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the sequence in the comparison window cancomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The terms “identical” or “identity”, in the context of two or morenucleic acids or polypeptide sequences, refer to two or more sequencesor subsequences that are the same. Sequences are “substantiallyidentical” to each other if they have a specified percentage ofnucleotides or amino acid residues that are the same (e.g., at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% identity over a specified region), when compared and aligned formaximum correspondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. These definitions also refer tothe complement of a test sequence. Optionally, the identity exists overa region that is at least about 50 nucleotides in length, or moretypically over a region that is 100 to 500 or 1000 or more nucleotidesin length.

The terms “similarity” or “percent similarity”, in the context of two ormore polypeptide sequences, refer to two or more sequences orsubsequences that have a specified percentage of amino acid residuesthat are either the same or similar as defined by a conservative aminoacid substitutions (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%,85%, 90%, or 95% similar over a specified region), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection.Sequences are “substantially similar” to each other if they are at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, or at least 55% similar to each other. Optionally,this similarly exists over a region that is at least about 50 aminoacids in length, or more typically over a region that is at least about100 to 500 or 1000 or more amino acids in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters are commonly used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities or similarities for the test sequencesrelative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, for example, by the local homologyalgorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by thehomology alignment algorithm of Needleman and Wunsch (J. Mol. Biol.48:443, 1970), by the search for similarity method of Pearson and Lipman(Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerizedimplementations of these algorithms (e.g., GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

Algorithms suitable for determining percent sequence identity andsequence similarity are the BLAST and BLAST 2.0 algorithms, which aredescribed in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977), andAltschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Softwarefor performing BLAST analyses is publicly available through publiclyavailable online and internet databases and the National Center forBiotechnology Information within the National Library of Medicine of theU.S. National Institutes of Health (http://www.ncbi.nlm.nih.gov/).

Rational Design of Taq DNA Polymerase Mutants

As outlined above, many strategies have been developed to improvediscrimination of the polymerase chain reaction to selectively amplify aspecific nucleic acid sequence based on the identity of a singlenucleotide polymorphism, which in the past most often involvedmodifications introduced into the primer while using a naturallyoccurring DNA polymerase. The ability of DNA polymerases to discriminatebetween match and mismatch at the 3′-end of the primer nucleic acid islimited and varies greatly with the identity of the specific base pairspresent. An alternative strategy to improve the selectivity of PCRamplification is to alter the properties of the DNA polymerase toimprove discrimination between a primer that is a match versus one whichhas a terminal mismatch to the template nucleic acid. The presentinvention provides for DNA polymerase mutants having improved mismatchdiscrimination for base pairing at the 3′-terminus of the primer,leading to improved specificity of the ensuing amplification reaction.

The rhPCR method employs blocked-cleavable primers which must beunblocked by the action of RNase H2 before amplification can commence.The enzymatic unblocking step requires cleavage at a single internal RNAbase within the primer, which is typically positioned at the SNP site.RNase H2 cleaves the RNA at the 5′-side, leaving a primer with a3′-hydroxyl which is capable of priming PCR. Cleavage by RNase H2 occurswith high efficiency when the primer matches the template and with lowefficiency when a mismatch is present due to a SNP. Therefore matchtemplates are amplified with greater efficiency than mismatch templates.It is thought that the primary mechanism that permits amplification ofmismatched templates begins with alternative cleavage of the substrate(i.e., the blocked-cleavable primer) at the 3′-side of the RNA residue,leading to inappropriate priming when a mismatch is present, retentionof the RNA base in the primer, and conversion of the PCR product toprimer sequence, which then faithfully replicates as a match insubsequent PCR cycles. Fidelity of the rhPCR process could be improvedthrough improvements in the DNA polymerase which limit its ability toinitiate DNA synthesis from a primer having a 3′-RNA residue. Thepresent invention provides for DNA polymerase mutants having a reducedability to initiate DNA synthesis from 3′-RNA containing primers,leading to improved specificity of the ensuing amplification reaction.

The present invention includes novel DNA polymerase mutants havingimproved discrimination for base identity at the 3′-end of the primernucleic acid and/or DNA polymerase mutants having decreased primingefficiency from a 3′-RNA residue.

A novel design strategy was developed to rationally design DNApolymerase mutants having improved discrimination at the 3′-terminalbase of the primer compared to discrimination of the native DNApolymerase, limiting the ability to initiate DNA synthesis if a mismatchis present or if an RNA residue is present. The process described hereinemployed the Taq DNA polymerase as the parent enzyme; the approach canbe applied to other DNA polymerases, especially if crystal structure isknown. The design strategy includes a first component based upontheoretical analyses of biophysical, biochemical and genetic informationrelating to the native DNA polymerase and, to a lesser extent, relatedpolymerases which differ in amino acid sequence. The design strategyincludes a second component based upon molecular biological andbiochemical analyses of known genetically-engineered mutant polymerasesto assist as a guide in predicting the effects of novel mutations in anattempt to rationally engineer new properties into the mutantpolymerase, in this case to improve 3′-nucleotide discrimination.

In the first stage, the mechanism of Taq DNA polymerase enzymaticreaction based upon published mutational structure-activity-relationship(SAR) studies was analyzed and correlated with protein structure, whenknown, and predicted using molecular dynamic simulations when not known.The mechanism of enzyme catalysis has been described in the prior art(Patel, P. H., et al., J. Mol. Biol. 2001, 308:823-837; Li, Y. &Waksman, G., Protein Sci 2001, 10:1225-1233). Amino acid residueslocated at the C-terminus, from positions 424 to 832, are responsiblefor the primer extension catalytic activity of the protein. Taq DNApolymerase binds the primer-template duplex to form a binary complex.This allows an incoming substrate dNTP to bind in the pocket at the3′-end of the primer to form an open ternary complex. If the dNTP iscomplementary to the template nucleotide, the active site changesconformation where the α-helix made from residues 659 to 671 rotatestowards the site, and template base rotates towards the incoming dNTP,encouraging formation of a Watson-Crick base pair. This event “closes”the ternary complex, and brings the α-phosphate group of the dNTP closeto the primer 3′-OH group. The oxygen of this hydroxyl group makes anucleophilic attack on the phosphorus, forms a covalent bond andpyrophosphate is released. Taq DNA polymerase catalytic activityrequires the presence of magnesium ions, which are assumed to facilitatedeprotonation of the attacking hydroxyl group.

One criteria for rational design of Taq DNA polymerase mutants havingimproved 3′-nucleotide discrimination is to provide for novel polymeraseenzyme variants having normal or near-normal polymerase processivitycompared with the native DNA polymerase. For this reason, the first stepof analysis serves to narrow the sequence space of amino acids that areavailable for alteration that should not compromise core enzymaticfunctions. For example, residues D610, D785, and E786 form the catalyticcore. Their carboxylate groups are assumed to bind divalent metal ions,which in turn bind and stabilize the incoming dNTP and the terminalnucleotide of the primer. Mutations of these three essential residuesare likely to render the polymerase inactive. Mutant polymerases whichinclude alterations of residues D610, D785 and E786 were thereforeexcluded from consideration. Likewise, mutations that affect fidelity ofcomplementary base recognition, such as residues that facilitate open toclosed ternary complex formation at a complementary dNTP and thetemplate base, were excluded from consideration.

Additional criteria of the first stage of analysis were to identify thepolymerase amino acid residues in the vicinity of the 3′ terminalnucleotide of the primer. For this purpose, the atomic three-dimensionalstructures of Taq DNA polymerase that are available from prior art (Eom,S. H., et al., Nature 1996, 382:278-281; Li, Y., et al., EMBO J. 1998,17: 7514-7525; Doublié, S., et al., Structure 1999, 7:R31-R35; Li, Y.,et al., Protein Sci 1998, 7:1116-1123) were selected for analyses. Thestructures were downloaded from the Protein Data Bank (H. M. Berman, etal., Nucleic Acids Research 2000, 28: 235-242). The structures of PDB ID2KTQ and 3KTQ were thoroughly analyzed because they show the open andclosed ternary complex of the large fragment of Taq DNA polymeraseco-crystalized with primer and template nucleic acids (Li, Y., et al.,EMBO J. 1998, 17: 7514-7525). This structure shows the location of theprimer 3′-terminus at the active site and its interaction with key aminoacid residues (FIG. 1).

For structure visualization, the hydrogen atom attached to the 2′ carbonis replaced with a hydroxyl group for primers modified with a3′-ribonucleotide. Those amino acid residues that are in close proximityto the 2′ carbon of the nucleotide at the 3′ terminus of the primer wereselected for additional analysis. These amino acid residues are listedin the Table 2 and are most likely to interact with the primer3′-terminal nucleotide. Mutation at these sites may affect catalyticactivity of the polymerase when primer modifications, like OH, areattached to the 2′ carbon atom of the ribose.

TABLE 2 Chemical groups in close vicinity to the 2′ carbon of theterminal 3′ primer nucleotide. Distance from C2′ of the primer terminalresidue¹ Chemical group ≤0.35 nm dNTP to be added to primer 0.35-0.40 nmD785² 0.40-0.45 nm H784, V783 0.45-0.50 nm R573 0.50-0.60 nm E786² ¹Thedistances were measured in the PDB ID 2KTQ structure. ²Residues D785 andE786 are catalytic core residues.

A further aspect of this criterion relates to approaches to increasespecificity while retaining catalytic activity of the polymerase. Oneapproach to increasing specificity of Taq DNA polymerase would be todecrease the size of the binding pocket, so that a modified primer wouldnot fit within it. Any additional chemical group will increases thevolume of space occupied by the 3′ nucleotide. To align atoms foreffective catalysis and nucleophilic attack, the active site pocket mustbe flexible to accommodate additional atom(s), for example, the oxygenof the OH group of an RNA residue. The size of the active site can bedecreased by substitution of neighboring amino acids with larger aminoacids. Additional consideration is given to the amino acid propertiesand abilities of their side chains to engage in electrostatic and vander Waals interactions. Amino acid can be categorized into groups ofpositively charged (R, H, K), negatively charged (D, E), uncharged polar(S, T, N, Q), hydrophobic (A, V, I, L, M, F, Y, W), and special (C, G,P) side chains. Mutations within a group are conservative and are morelikely to maintain existing properties while mutations across groups orwithin the special group amino acids are more likely to result insubstantial changes of enzyme activity and/or specificity.

Another approach to increasing 3′-nucleotide discrimination of Taq DNApolymerase is to employ residue substitutions that decrease theflexibility of the binding pocket. Examples include substitution ofamino acid aliphatic side chains with aromatic side chains, which leadto a higher energetic barrier to change rotamer conformations. Asexplained above, the residues of the catalytic core are preferablyunaltered, residues spatially near the catalytic core are given thegreatest attention for change. For example, three non-catalytic coreresidues of Table 2, H784, V783, and R573 are herein proposed to besubstituted for larger or less flexible amino acids while themaintaining the general physical characteristics of their side chains.These residues exhibit key interactions with the ribose moiety of theprimer through a water-mediated hydrogen-bonding network. R573 alsobinds to the primer base in the minor groove of primer-template duplex.Mutants ID 1 to ID 4 were designed using this strategy (Table 3). Thenext mutant, ID 5, Q582K, was designed to alter interactions with andthe position of the important H784 residue. It is seen from the knowncrystal structure that Q582 is situated on the opposite side of H784from the oligonucleotide primer. Substitution of Q582H may shift H784towards the terminal primer nucleotide, leading to a more constrainedbinding pocket. The interactions of residue 582 with thenext-to-terminal nucleotide may also be affected.

Residues that stack above the incoming dNTP molecule can also influencethe size of the binding pocket in the polymerase active site. Forexample, substitutions at F667, which is located at this position, areknown to change selectivity towards the incoming dNTP. For example, theF667Y substitution significantly improves incorporation ofdideoxynucleoside triphosphates by Taq DNA polymerase (Tabor, S. &Richardson, C. C., Proc. Nat. Acad. Sci. USA 1995, 92:6339-6343), auseful property for DNA polymerases employed in Sanger method terminatorDNA sequencing. Mutant ID 6 increases the size of the aromatic sidechain of F667 from phenylalanine to tryptophan, in an attempt to pushthe dNTP against the primer terminal ribonucleotide and decrease abilityof binding pocket to accommodate a 2′ hydroxyl group, thereby biasingthis mutant against primers containing a 3′-RNA residue. Mutant ID7 wasdesigned based on similar conceptual framework. The H639 interacts withF667 amino acid and H639W mutant might also push F667 towards theincoming dNTP.

Additional mutations were considered that can effectively reduce thebinding pocket size of the polymerase. Mutants IDs 8 to 16 were designedfrom a negative inferential analysis based on published studies of“relaxed specificity” mutant polymerases. Mutations have been reportedthat can modify polymerase specificity towards the ribose of theincoming dNTP. The prior art Taq DNA polymerase variants described wereevolved from large random libraries either through selection orscreening. Chen et al. described mutations that allow Taq DNA polymeraseto incorporate a dNTP with large substituents on the ribose 3′ carbonatom (Chen, F., et al., Proc. Nat. Acad. Sci. USA 2010, 107:1948-1953).This residue was found to be important because it also interacts withF667. The substitution L616A decreases specificity by giving more spaceto the phenylalanine residue. Mutant ID 8 (L616M) was designed toproduce the opposite effect. The methionine substitution may subtlyincrease the steric constraints at this site compared to leucine. Thisrestriction may make the active site less likely to accommodate extrasubstituents in a dNTP or in a primer nucleotide, which could reduceactivity of 3′-RNA containing primers or those having a mismatch totemplate, which presumably occupies more space than primers having aperfect match to the template nucleic acid.

A similar conceptual framework was applied to design Taq DNA polymerasemutant ID 9. Mutations I614E, E615G were reported to relax the activesite pocket, so that the polymerase could extend a primer using2′-O-methyl ribonucleoside triphosphates (Fa, M., et al., J. Am. Chem.Soc. 2004, 126:1748-1754). The nature of these mutations is essentiallythe shift of glutamic acid from residue 615 to 614. A shift in theopposing direction, E615L, L616E may therefore impose constraints on theactive site and produce a Taq DNA polymerase mutant that will not acceptribonucleotide residues.

Another approach to increasing 3′-nucleotide discrimination is to focuson sites of interest identified in Taq DNA polymerase studies thatreported amino acid substitutions which increased base selectionfidelity and decreased incorporation of mispaired base pairs (i.e.,those mutation that improve replication fidelity). These changes couldpotentially affect selectivity of Taq DNA polymerase regarding tomodifications of terminal primer nucleotide as well. One location thatwas reported to improve fidelity involves the F667 residue andneighboring amino acids (Suzuki, M., et al., J. Biol. Chem. 2000,275:32728-32735). Another site of potential interest includes residues782 to 784, adjacent to an essential aspartic acid residue (Strerath,M., et al., ChemBioChem 2007, 8:395-401). Mutants ID 10 to ID 13 weredesigned to alter amino acid character at these positions. F667 affectsspecificity as it interacts with the terminal base of the primer andstacks on the base of the incoming dNTP; this residue resides in theO-helix. Residues I665 and A661 are located on the opposite side of thehelix. Mutation here to larger amino acids (A661E,I665W) may move theO-helix towards the active site, restricting the size of the activepocket and limiting ability of the polymerase to accept mispaired basesor RNA residues (Mutant ID 10: A661E,I665W,F667L).

Data derived from mutagenesis studies of different polymerases can alsobe used to help select positions for modification, but use of this datais more difficult in the absence of crystal structure or due to possibledifferences in effects between the polymerases. Polymerase I fromEscherichia coli (“E. coli Pol”) shows a somewhat similar structure atthe active site when compared to Taq DNA polymerase and maintainsidentical essential catalytic residues. Both protein sequences exhibithigh degree of homology (Li, Y., et al., EMBO J. 1998, 17: 7514-7525).Thus, mutations reported for Escherichia coli DNA polymerase were alsoconsidered, by extrapolating amino acid position to the correspondingpositions in the Taq DNA polymerase. For example, the triplet amino acidsubstitutions, Q879P, V880L, H881Q, improved base fidelity of E. coliDNA polymerase (Summerer, D., et al., Angew. Chem. Int. Ed. 2005,44:4712 -4715). Substitutions in mutant ID 14 includes substitutions atQ782, V783, H784 in the Taq DNA polymerase active site, which appear tocorrespond to this E. coli residue triplet.

A number of additional substitutions in the E. coli DNA polymerase areknown which decrease or increase the specificity of primer extension(Minnick, D. T., et al., J. Biol. Chem. 1999, 274:3067-3075). MutantsQ849A and R754A improved fidelity. These have locations equivalent toQ754 and R659 in the Taq DNA polymerase active site, respectively.Arginine 659 has a significant impact on selection of the basecomplementary to the template base. This appears to be general featurein the polymerase A family. For example, in Thermotoga neapolitanapolymerase I, the equivalent residue is R722. Mutation of this residueto histidine increases fidelity of this polymerase (Yang, S. W., et al.,Nucleic Acids Res. 2002, 30:4314-4320). These two residues were alsoselected for study (mutants ID 15 and 16 of Table 3). Mutant ID 17represents combination of the mutations studied in Mutants ID 2 and 3.Mutant ID 18 represents a modification of triple mutant ID 14 (Q782P,V783L, H784Q) reduced to a double mutant (V783L, H784Q) by eliminatingthe Q782P mutation; the substitution of a less flexible P for Q residuewill likely cause significant structural perturbation which would alterfunction, and Mut ID 18 may avoid this problem. Initial testingindicated that more than one mutant at position H784 showed improvedmismatch discrimination, suggesting that this position was generallyimportant for determining primer specifcity. Therefore a comprehensivestudy of amino acid substitutions at this site was performed, comprisingMut IDs 19-36.

TABLE 3 Novel Taq DNA polymerase mutants selected for study. Specificamino acid changes from Mutant ID sequence in Table I 1 V783I 2 V783F 3H784Q 4 R573H 5 Q582K 6 F667W 7 H639W 8 L616M 9 E615L, L616E 10 A661E,I665W, F667L 11 Q782I, H784F 12 Q782I, V783L, H784L 13 Q782S, V783F,H784N 14 Q782P, V783L, H784Q 15 Q754A 16 R659H 17 V783F, H784Q 18 V783L,H784Q 19 H784G 20 H784A 21 H784S 22 H784T 23 H784C 24 H784V 25 H784L 26H784I 27 H784M 28 H784P 29 H784F 30 H784Y 31 H784W 32 H784D 33 H784E 34H784N 35 H784K 36 H784R

The second component of the design strategy includes molecularbiological and biochemical analyses of genetically-engineered Taq DNApolymerase mutants to identify novel enzymes having improved3′-nucleotide discrimination. This requires expression of native Taq DNApolymerase and the series of designed mutants in a suitable host, suchas the bacterium E. coli. To maximize expression, the codons of thenative gene sequence encoding Taq DNA polymerase were altered andoptimized for expression in E. coli using standard codon usage tablesfor this organism (see: Codon usage tabulated from the international DNAsequence databases: status for the year 2000. Nakamura, Y., Gojobori, T.and Ikemura, T. (2000) Nucleic Acids Res. 28:292). Codon optimizationdoes not alter the amino acid sequence of the expressed protein. Arecombinant form of a codon-optimized gene encoding the unaltered TaqDNA polymerase peptide was assembled and cloned into a plasmid vector asan artificial gene made from synthetic oligonucleotides using standardmethods (Example 1). The plasmid vector for this purpose can be anyplasmid vector routinely available in the art. Synthetic recombinantforms of the series of identified desired Taq DNA polymerase mutants(Table 3, Mutant IDs 1-36) were prepared by site directed mutagenesis ofthe previously assembled codon-optimized recombinant native Taq DNApolymerase as the substrate for site directed mutagenesis (SDM), usingtechniques well known to those with skill in the art (Example 1). Theunmodified and mutant Taq DNA polymerases were prepared from E. colihost cells following introduction of expression vectors that contain thecorresponding recombinant forms of the genes operably linked to suitabletranscriptional and translational control elements.

The enzymatic properties of the unmodified Taq DNA polymerase and mutantTaq DNA polymerases were evaluated for primer extension assays,thermostability, PCR assays, allele-specific PCR assays, ability toemploy primers having a 3′-ribonucleotide, as well as their suitabilityfor use in rhPCR assays. The mutant Taq DNA polymerases displayed one offour categories of enzymatic properties: (1) inactivated polymeraseactivity; (2) normal polymerase activity; (3) improved 3′-nucleotidediscrimination activity, but having reduced activity (for example,reduced processivity); and (4) improved 3′-nucleotide discrimination andhaving normal or near normal polymerase activity (for example,processivity comparable to the native polymerase).

Mutant Taq DNA polymerases having the fourth category of enzymaticproperties displayed comparable or enhanced 3′-mismatch discrimination(that is, comparable or improved performance in standard primerextension assays and allele-specific PCR assays when compared to thewild-type Taq DNA polymerase); enhanced 3′-nucleotide discrimination(that is, reduced primer extension activity from templates containingRNA-containing primers when compared to the wild-type Taq DNApolymerase) and enhanced rare allele discrimination (for example,improved specificity in rhPCR assay when compared to the wild-type TaqDNA polymerase). These mutant Taq DNA polymerases include mutations atone of the following residue position(s): (1) A661E; I665W; F667L triplesubstitution mutant peptide (Mutant ID 10 of Table 3); (2) V783F singlesubstitution mutant peptide (Mutant ID 2 of Table 3); H784Q singlesubstitution mutant peptide (Mutant ID 3 of Table 3); and V783L; H784Qdouble substitution mutant peptide (Mutant ID 18 of Table 3), H784A,single substitution mutant peptide (Mutant ID 20 of Table 3); H784S,single substitution mutant peptide (Mutant ID 21 of Table 3); H784T,single substitution mutant peptide (Mutant ID 22 of Table 3); H784V,single substitution mutant peptide (Mutant ID 24 of Table 3); H784I,single substitution mutant peptide (Mutant ID 26 of Table 3); H784M,single substitution mutant peptide (Mutant ID 27 of Table 3); H784F,single substitution mutant peptide (Mutant ID 29 of Table 3); and H784Ysingle substitution mutant peptide (Mutant ID 30 of Table 3).

Thus, the novel design algorithm provides a robust approach to predictmutant DNA polymerases having improved 3′-nucleotide discrimination, asadjudged by their activity in allele-specific PCR, rare allele detectionassays and rhPCR assays that utilize templates with or without a 3′-RNAresidue in the primer. Specifically, residues V783 and H784 wereidentified as critical residues which influence the ability of thepolymerase to interrogate the status of the 3′-base of the primeroligonucleotide (e.g., whether this residue is matched or mismatchedwith template and/or whether this residue is DNA or RNA). Thesignificance of these residues in polymerase function was heretofore notappreciated. In addition to the mutations directly testing in theexample, the present invention also contemplates other amino acidsubstitutions at these two positions, or double-mutants affecting boththe V783 and H784 sites.

The properties of these mutants are further described in the Examplespresented herein. Importantly, however, the design strategy employedherein enables access to functional space for novel Taq DNA polymerasemutants that were previously unrecognized or predicted or otherwise notobtained using other approaches (for example, phylogenetic comparativeanalysis or earlier attempts using random mutagenesis).

Evaluation of Taq DNA Polymerase Mutants at Residue Positions 783 and784

The present disclosure demonstrates that mutation at residue positions783 and/or 784 results in active Taq DNA polymerase mutants havingenhanced template discrimination activity, as compared to unmodified TaqDNA polymerase. Thus, the entirety of the sequence space that includesevery conceivable single amino acid substitution at the individualpositions 783 or 784 as well as every conceivable double amino acidsubstitution at both positions 783 and 784 fall within the scope of thepresent disclosure as related to Taq DNA polymerase. Accordingly, thoseactive Taq DNA polymerase mutants selected from the mutant sets of 19single residue 783 mutants, 19 single residue 784 mutants (Table 3 MutIDs 19-30) and 361 double residue 783/784 mutants that also possessenhanced template discrimination activity fall within the scope of thepresent disclosure.

Because Taq DNA polymerase is a thermostable enzyme, one facile approachto screening the candidate collection of 399 single- anddouble-substitution mutants at residue positions 783 and 784 is toperform a PCR assay with a pre-treated sample encoding a candidate TaqDNA polymerase mutant enzyme. The sample can be a selected individualcolony or corresponding micro-cultures (for example, 50 μL to 1.0 mLcultures) obtained from the individual colony transformed withrecombinant DNA that expresses a desired recombinant Taq DNA polymerasemutant gene. The pre-treatment regimen can include the step ofpre-incubating the sample at 70-95° C., followed by the step ofclarifying the supernatant to remove the denatured cellular debris. Forsamples that express thermostable polymerase activity under standard PCRassay conditions, the corresponding recombination DNA can be furthercharacterized to confirm the sequence of the desired recombinant Taq DNApolymerase mutant genotype and the polymerase protein purified foradditional biochemical analysis. For the purposes of this disclosure, aTaq DNA polymerase mutant that expresses thermostable polymeraseactivity at a level of at least 0.01 of that expressed by wild-type TaqDNA polymerase under comparable PCR assay conditions can be adjudged aspossessing thermostable polymerase activity.

Evaluation of Other Select Polymerase Candidate Mutants FunctionallyHomologous to Taq DNA Polymerase at Residue Positions 783 and 784

Comparative phylogenetic analysis tools can be used to identify thesequence space of other thermoactive polymerases having homologoussequence information relative to the unmodified Taq DNA polymerase atresidue positions corresponding to V783 and H784. As explained supra, astrong prediction of the comparative phylogenetic analysis is thatstructural sequences shared among DNA polymerases acrossphylogenetically diverse species are conserved for functional reasons.If the identified V783/H784 residues of Taq DNA polymerase are invariantin sequence identity among wild-type polymerases from diverse species,that observation strongly supports the conclusion that nature selectedagainst the specific variation of amino acid substitutions at thosepositions that result the observed enhanced template discriminationactivity of the engineered Taq DNA polymerase mutants disclosed herein.

Example 11 provides an exemplary BLAST search using Taq DNA polymerasesequences encompassing positions V783 and H784 as a comparison window toidentify candidate wild-type DNA polymerases from other species sharingextensive sequence identity with Taq DNA polymerase. As furtherelaborated in Example 11, the BLAST results revealed that virtuallyevery identified DNA polymerase from diverse species has maintained Valand His at positions orthologous to V783 and H784 of Taq DNA polymerase.Thus, the BLAST results confirm a natural counter-selection against DNApolymerases having enhanced template discrimination activity and providestrong evidence that the disclosed engineered Taq DNA polymerase mutantshaving these properties are novel and non-obvious. Like that observedwith the engineered Taq DNA polymerase mutants, each of the identifiednon-Taq DNA polymerases represent a sequence space from which engineeredmutant enzymes can be generated having enhanced template discriminationactivity, as compared to their respective unmodified counterparts.

In those cases where comparative phylogenetic analysis cannot access thesequence space of more evolutionary distant organisms, a comparativebiophysical crystallographic analysis can provide clues to the relevantsequence residues having functional homology to Taq DNA polymeraseresides V783 and H784. As explained supra, the Q782, V783 and H784residue triplet of Taq DNA polymerase was selected for analysis basedupon the corresponding triplet amino acid substitutions Q879P, V880L andH881Q of E. coli DNA polymerase having improved base fidelity and asimilar active site architecture to that of Taq DNA polymerase.Conversely, based upon the noted enhanced template discriminationactivity of V783 and H784 Taq DNA polymerase mutants relative towild-type Taq DNA polymerase, the present disclosure contemplates thatthe corresponding substitutions at V880 and H881 of the E. coli DNApolymerase will possess enhanced template discrimination activityrelative to wild-type E. coli DNA polymerase.

Identification and Characterization of Non-VH-Related Polymerase Mutantshaving Enhanced Template Discrimination Activity.

The foregoing collection of DNA polymerases share extensively conservedsequences in the region corresponding to V783 and H784 of Taq DNApolymerase (“VH-related polymerases”). Comparative biophysical analysisis useful for identifying wild-type DNA polymerases having differentamino acid sequences in the functionally homologous positions as V783and H784 of Taq DNA polymerase (“non-VH-related DNA polymerases”). Theinstant disclosure contemplates engineering mutant polymerases havingenhanced template discrimination activity from these non-VH-related DNApolymerases in the same manner as disclosed for the VH-related DNApolymerases. Candidate non-VH resides for directed mutagenesis andanalysis by enhanced template discrimination activity assays includethose resides within 0.40-0.45 nm of the C2′ of the primer terminalresidue, as revealed in the polymerase:template co-crystal structure.

Combination of Site-Specific Taq DNA Mutants with Deletion of the5′-Exonuclease Domain.

The present invention discloses novel Taq DNA Polymerase mutants thatshow improved discrimination of mismatches positioned at the 3′-residueof the primer oligonucleotide and/or discrimination against the presenceof an RNA residue at the 3′-end of the primer oligonucleotide. Improvedmismatch discrimination has been described for the “KlenTaq” deletionmutant of Taq DNA Polymerase, which entirely removes the domain having5′-exonuclease activity (Barnes, W. M., Gene 112:29-35, 1992).Combination of the novel mutants of the present invention with theKlenTaq 5′-exonuclease domain deletion led to further improvements inmismatch discrimination (Examples 18-22), however this comination led todecreases in enzymatic activity which may reduce utility of this familyof double-mutants. In some circumstances, particularly when ampliconsize is small and limited processivity could be tolerated, the enhanceddecrimination of these mutants will have benefit.

Reaction Mixtures

In another aspect, reaction mixtures are provided comprising thepolymerases with increased 3′-nucleotide discrimination activity. Thereaction mixtures can further comprise reagents for use in, for example,nucleic acid amplification procedures (for example, PCR, RT-PCR, rhPCR),DNA sequencing procedures, or DNA labeling procedures. For example, incertain embodiments, the reaction mixtures comprise a buffer suitablefor a primer extension reaction. The reaction mixtures can also containa template nucleic acid (DNA and/or RNA), one or more primer or probepolynucleotides, nucleoside triphosphates (including, for example,deoxyribonucleotides, ribonucleotides, labeled nucleotides,unconventional nucleotides), salts (for example, Mn²⁺, Mg²⁺), and labels(for example, fluorophores). In some embodiments, the reaction mixturefurther comprises double stranded DNA binding dyes, such as SYBR green,or double stranded DNA intercalating dyes, such as ethidium bromide. Insome embodiments, the reaction mixtures contain a 5′-sense primerhybridizable, under primer extension conditions, to a predeterminedpolynucleotide template, or a primer pair comprising the 5′-sense primerand a corresponding 3′-antisense primer. In certain embodiments, thereaction mixture further comprises a fluorogenic FRET hydrolysis probefor detection of amplified template nucleic acids, for example a Taqman®or PrimeTime® probe. In some embodiments, the reaction mixture containstwo or more primers that are fully complementary to single nucleotidepolymorphisms or multiple nucleotide polymorphisms. In some embodiments,the reaction mixtures contain alpha-phosphorothioate dNTPs, dUTP, dITP,and/or labeled dNTPs such as, for example, fluorescein- or cyanin-dyefamily dNTPs. In some embodiments, the reaction mixtures containblocked-cleavable primers and RNase H2.

Kits

In another aspect, kits are provided for use in primer extension methodsdescribed herein. In some embodiments, the kit is compartmentalized forease of use and contains at least one container providing a DNApolymerase of the invention having increased 3′-nucleotidediscrimination in accordance with the present disclosure. One or moreadditional containers providing additional reagent(s) can also beincluded. Such additional containers can include any reagents or otherelements recognized by the skilled artisan for use in primer extensionprocedures in accordance with the methods described above, includingreagents for use in, for example, nucleic acid amplification procedures(for example, PCR, RT-PCR, rhPCR), DNA sequencing procedures, or DNAlabeling procedures. For example, in certain embodiments, the kitfurther includes a container providing a 5′-sense primer hybridizable,under primer extension conditions, to a predetermined polynucleotidetemplate, or a primer pair comprising the 5′-sense primer and acorresponding 3′-antisense primer. In some embodiments, the kit includesone or more containers containing one or more primers that are fullycomplementary to single nucleotide polymorphisms or multiple nucleotidepolymorphisms, wherein the primers are useful for multiplex reactions,as described above. In some embodiments, the reaction mixtures containone or more containers containing blocked-cleavable primers. In someembodiments, the reaction mixtures contain one or more containerscontaining RNase H2. In other, non-mutually exclusive variations, thekit includes one or more containers providing nucleoside triphosphates(conventional and/or unconventional). In specific embodiments, the kitincludes alpha-phosphorothioate dNTPs, dUTP, dITP, and/or labeled dNTPssuch as, for example, fluorescein- or cyanine-dye family dNTPs. In stillother, non-mutually exclusive embodiments, the kit includes one or morecontainers providing a buffer suitable for a primer extension reaction.In some embodiments, the kit includes one or more labeled or unlabeledprobes. Examples of probes include dual-labeled FRET (fluorescenceresonance energy transfer) probes and molecular beacon probes. Inanother embodiment, the kit contains an aptamer, for example, for hotstart PCR assays.

The present disclosure contemplates kits that provide novel DNApolymerases having enhanced template discrimination activity. Asdemonstrated in more detail in the examples, each DNA polymerase candisplay a unique signature of enhanced template discrimination activity.Certain DNA polymerases can display a relatively greater 3′-nucleotidediscrimination, as compared to its other activities (3′-mismatchdiscrimination and rare allele discrimination), while other DNApolymerases can display a relatively greater 3′-mismatch discrimination,as compared to its other activities (3′-nucleotide discrimination andrare allele discrimination), and yet other DNA polymerases can display arelatively greater rare allele discrimination, as compared to its otheractivities (3′-nucleotide discrimination and 3′-mismatchdiscrimination). Accordingly, kits can include individual containers ofspecific DNA polymerases having an activity profile optimally tailoredto a specific enhanced template discrimination activity for a specificassay platform. Alternatively, kits can include a single container thatincludes a plurality of DNA polymerases having an activity profileoptimally tailored to accommodate enhanced template discriminationactivity as may be needed for a plurality of assay platforms.

EXAMPLES

The present invention is further illustrated by reference to thefollowing Examples. However, it should be noted that these Examples,like the embodiments described above, are illustrative and are not to beconstrued as restricting the enabled scope of the invention in any way.

Example 1 Cloning and Expression of a Codon Optimized DNA Polymerasefrom Thermus aquaticus

The amino acid and gene sequences for Taq DNA polymerase are known(Table 1, SEQ ID NOs. 1 and 2). Because codon usage differs amongorganisms, the codons of the native gene sequence encoding Taq DNApolymerase were optimized for expression in E. coli using standard codonusage tables (see: Codon usage tabulated from the international DNAsequence databases: status for the year 2000. Nakamura, Y., Gojobori, T.and Ikemura, T. (2000) Nucleic Acids Res. 28:292); synonymous codonchanges were introduced to avoid repeated use of identical codons over a20 amino acid stretch. A recombinant codon-optimized gene encoding theTaq DNA polymerase unmodified peptide was assembled from syntheticoligonucleotides using standard methods. The gene was made in threefragments, each of which was subcloned in a plasmid vector; sequencesare shown in Table 4 (SEQ ID NOs. 3-5). Sequence identity was verifiedby Sanger DNA sequencing. The three Taq DNA polymerase subfragments wereassembled together using the Gibson assembly method (Gibson, D. G. etal. Nature Methods, 343-345 (2009)) and cloned into a the plasmidexpression vector pET-27b(+) using terminal Nde I and Not I restrictionssites to create a final, full-length codon optimized Taq DNA polymerasegene (designated “OptiTaq”). Sequence was verified by Sanger DNAsequencing; sequence is shown in Table 4 (SEQ ID NO. 6). The translatedamino acid sequence of the new codon optimized gene is identical tonative Taq DNA polymerase (Table 1, SEQ ID NO. 1).

TABLE 4DNA sequence of Tag DNA Polymerase codon-optimized for expression inE. coli. Name Sequence SEQ ID NO. 3CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATaq subfragmentTCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGG #1TCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGT SEQ ID NO. 4TCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTaq subfragmentTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGC #2AGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAAC SEQ ID NO. 5CACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCTaq subfragmentAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAA #3GGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGGTCCATGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC SEQ ID NO. 6 CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCA Complete codon-TCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGoptimized TaqTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGDNA polymeraseGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAA “OptiTaq”GGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGGTCCATGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC For the final completed “OptiTaq” clone, NdeI and Not I restrictions sites are underlined. The ATG start codon isidentified in bold font.

Example 2 Production of Codon Optimized Taq DNA Polymerase Mutants

Eighteen mutant versions of Taq DNA polymerase (Table 3, Mut IDs 1-18)were made by site directed mutagenesis of the cloned OptiTaqcodon-optimized Taq DNA polymerase. Specific mutations were introducedinto the OptiTaq sequence using the method of PCR site-directedmutagenesis (Weiner M P, et al., Gene. 151(1-2):119-23 (1994)). Eachmutagenesis reaction employed 10 pmoles of two complementaryoligonucleotides (Table 5) containing the desired base changes, annealedto the double-stranded OptiTaq plasmid (20 ng), 5 U KOD DNA polymerase(Novagen-EMD Chemicals, San Diego, Calif.), 1.5 mM MgSO₄, in 1× KOD PCRbuffer. Thermal cycling parameters were 95° C. for 3 minutes (95° C. for20 sec-55° C. for 20 sec-70° C. for 2.5 minutes) for 16 cycles followedby a 70° C. soak for 4 minutes. After PCR site-directed mutagenesis, theamplified product was treated with 10 U of Dpn I (NEB, Ipswich, Mass.),at 37° C. for 1 hour, followed by inactivation at 80° C. for 20 minutes.1/110^(th) of the digestion material was transformed into XL-1 Bluecompetent bacteria. Bacterial clones were isolated, plasmid DNAprepared, and individual mutations were confirmed by Sanger DNAsequencing. All mutants remained in the pET-27b(+) expression vector,which is suitable for expressing the recombinant proteins in E. coli.

TABLE 5 Oligonucleotides used for site-directed mutagenesis to produce18 Tag DNA Polymerase mutants. Amino Sequence″ Sequence″ Mutant acidSense mutagenesis SEQ Antisense mutagenesis SEQ ID changesoligonucleotide ID No. oligonucleotide ID No. 1 V783Iaatgggcgcacgtatgcttct 7 gggcttctaacaccagctcgtca 8 gcagATTcatgacgagctggttgAATctgcagaagcatacgtgc gttagaagccc gcccatt 2 V783Faatgggcgcacgtatgcttct 9 gggcttctaacaccagctcgtca 10 gcagTTCcatgacgagctggttgGAActgcagaagcatacgtgc gttagaagccc gcccatt 3 H784Qgggcgcacgtatgcttctgca 11 taggggcttctaacaccagctcg 12ggtcCAGgacgagctggtgtt tcCTGgacctgcagaagcatacg agaagccccta tgcgccc 4R573H caaccagacggcgactgcaac 13 ggagatttggatccgagctagac 14cggcCATctgtctagctcgga agATGgccggttgcagtcgccgt tccaaatctcc ctggttg 5Q582K tctgtctagctcggatccaaa 15 ccaagggtgtacggaccggaatg 16tctcAAAaacattccggtccg ttTTTgagatttggatccgagct tacacccttgg agacaga 6F667W gcgccgtgcagctaaaacaat 17 gagcgctcattccgtacagcact 18taatTGGggagtgctgtacgg ccCCAattaattgttttagctgc aatgagcgctc acggcgc 7H639W cgtgtttcaagaggggcgtga 19 cgaacatccatgaggcagtttct 20tattTGGacagaaactgcctc gtCCAaatatcacgcccctcttg atggatgttcg aaacacg 8L616M cgcattggactactcgcagat 21 caccagagagatgtgcgaggacg 22tgagATGcgcgtcctcgcaca cgCATctcaatctgcgagtagtc tctctctggtg caatgcg 9E615L ggtcgcattggactactcgca 23 caccagagagatgtgcgaggacg 24 L616EgattCTGGAGcgcgtcctcgc cgCTCCAGaatctgcgagtagtc acatctctctggtg caatgcgacc10 A661E cgtgaagcagtggatcctttg 25 gcgatgagcgctcattccgtaca 26 I665WatgcgccgtGAAgctaaaaca gcactccCAAattCCAtgtttta F667LTGGaatTTGggagtgctgtac gcTTCacggcgcatcaaaggatc ggaatgagcgctcatcgccactgcttcacg 28 11 Q782I ggaaatgggcgcacgtatgct 27taggggcttctaacaccagctcg H784F tctgATCgtcTTCgacgagcttcGAAgacGATcagaagcatacg ggtgttagaagccccta tgcgcccatttcc 12 Q782Iggaaatgggcgcacgtatgct 29 taggggcttctaacaccagctcg 30 V783LtctgATTTTGCTGgacgagct tcCAGCAAAATcagaagcatacg H784L ggtgttagaagcccctatgcgcccatttcc 13 Q782S ggaaatgggcgcacgtatgct 31 taggggcttctaacaccagctcg32 V783F tctgTCCTTCAACgacgagct tcGTTGAAGGAcagaagcatacg H784Nggtgttagaagccccta tgcgcccatttcc 14 Q782P ggaaatgggcgcacgtatgct 33taggggcttctaacaccagctcg 34 V783L tctgCCGTTACAGgacgagcttcCTGTAACGGcagaagcatacg H784Q ggtgttagaagccccta tgcgcccatttcc 15 Q754Agcgtatggcatttaatatgcc 35 gtttcatgaggtcagctgcagta 36tgtaGCGggtactgcagctga ccCGCtacaggcatattaaatgc cctcatgaaac catacgc 16R659H acgtgaagcagtggatccttt 37 caaaattaattgttttagctgca 38gatgCACcgtgcagctaaaac cgGTGcatcaaaggatccactgc aattaattttg ttcacgt 17V783F aatgggcgcacgtatgcttct 39 GcttctaacaccagctcgtcCTG 40 H784QgcagTTCCAGgacgagctggt GAActgcagaagcatacgtgcgc gttagaagc ccatt 18 V783Laatgggcgcacgtatgcttct 41 gggcttctaacaccagctcgtcC 42 H784QgcagCTGCAGgacgagctggt TGCAGctgcagaagcatacgtgc gttagaagccc gcccatt DNAbases identical to codon optimized OptiTaq are shown in lower case;those specific for the mutations introduced by site-directed mutagenesisare shown in upper case.

Example 3 Expression of Recombinant Taq DNA Polymerases

The following example demonstrates the expression of recombinant Taq DNApolymerase unmodified and mutant peptides. The synthetic gene sequencesfrom Examples 1, 2 and 12 were cloned in the pET-27b(+) expressionvector (Novagen, EMD Biosciences, La Jolla, Calif.). This vector placessix histidine residues (which together comprise a “His-tag”) at thecarboxy terminus of the expressed peptide, followed by a stop codon. A“His-tag” permits use of rapid, simple purification of recombinantproteins using Ni²⁺ affinity chromatography methods which are well knownto those with skill in the art. Alternatively, the synthetic genes couldbe expressed in native form without the His-tag and purified using sizeexclusion chromatography, anion-exchange chromatography, or other suchmethods, which are also well known to a person of ordinary skill in theart.

BL21(DE3) competent E. coli cells (Novagen) were transformed with ˜1 ngof each plasmid. Briefly, plasmids were added to the cells on ice andgently stirred. After a 5 minute incubation on ice, cells were heatshocked at 42° C. for 30 seconds, then returned to ice for 2 minutes.Room temperature SOC (80 μL) was added to the transformed cells,followed by a 1 hour outgrow period at 37° C., with agitation at 250rpm. Cells were plated (20 μL) on 37° C. pre-warmed LB/Kan plates (LuriaBroth agar plates supplemented with 50 μg/mL kanamycin) and were placedat 37° C. overnight. The next morning, one colony was picked and grown(37° C., 250 rpm) in 10 mL LB/Kan broth (50 μg/mL) to log phase (OD6000.3-0.9). Cells were then induced with Overnight Express™ AutoinductionSystem 1 (Novagen) in Terrific Broth at 37° C., 250 rpm following theprotocol recommended by the manufacturer. Culture volumes were 100 mLfor wild type OptiTaq and 200 mL for mutants. Growth saturation wasreached after 18 hours, and the culture was pelleted at 10,000×g for 10minutes in a Beckman Avanti™ J-25 Centrifuge. The pellet (˜6 g) waslysed using 30 mL BugBuster® Protein Extraction Reagent (Novagen), 30 kUrLysozyme™ Solution (Novagen) and 1500 U DNase I (Life Technologies,Grand Island, N.Y.) to release soluble proteins and degrade nucleicacids according to the manufacturer's instructions. Followingcentrifugation at 15,000×g for 20 minutes to remove cell debris, thelysates were heated at 75° C. for 15 minutes to inactive DNase I andother cellular nucleases. The lysates were then spun at 15,000×g for 20minutes to sediment denatured protein. The heat denaturation andcentrifugation steps provide significant purity enrichment of therecombinant enzymes. Both “total” and “soluble” fractions of thebacterial lysates were analyzed using SDS 4-20% polyacrylamide gelelectrophoresis for 1 hour at 125 V. Proteins were visualized withCoomassie Blue staining for 1 hour, followed by 3-4 rounds of destaininguntil protein bands were clear.

The recovered soluble protein was passed over a Ni²⁺ affinity columncontaining His⋅Bind Resin (Novagen) and eluted with a buffer containing200 mM imidazole (200 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH7.9). The purified protein (˜6 mL) was then concentrated at 3210×g in aBeckman Coulter 6R tabletop centrifuge swinging bucket rotor using anAmicon Ultra-15, PLGC Ultracel-PL Membrane, 10 KDa concentrator (EMDMillipore, Billerica, Mass.) to ˜200 μL and stored at −20° C. untildialysis. The concentrated protein was then dialyzed against storagebuffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50%glycerol, 0.1% Triton X-100) at 4° C. overnight, followed by 3×2 hours(at 1000 fold ratio of protein solution to dialysis buffer each time).The final purified protein was stored at −20° C. Using this protocol,100 mL of an autoinduced culture yielded ˜1.2 mg/67.6 μM/12,168 pmol ofpurified soluble protein for OptiTaq. Similar yields were obtained forthe mutant DNA polymerases.

To determine protein concentration, samples were examined alongsideknown quantities of BSA (bovine serum albumin) using SDS 4-20%polyacrylamide gel electrophoresis for 1 hour. Proteins were visualizedwith Coomassie Blue staining for 1 hour, followed by 3-4 rounds ofdestaining until protein bands were clear. Band intensity was analyzedusing ImageJ software (National Institutes of Health, Bethesda, Md.).

To evaluate purity and quality of the recombinant protein preparations,500 ng of each recombinant protein (wild type OptiTaq and each mutant)were separated on a 4-20% SDS-PAGE gel, stained with Coomassie Blue, andvisualized. The recombinant proteins all migrate at the appropriateposition on the gel for proteins having a molecular weight of 97.1 kDa.The preparations show relatively high purity with few additional speciesdetected. Gel images are shown in FIGS. 2A, 2B, 2C, and 2D. Similar gelswere run for MUT IDs 22 (H784T), 24 (H784V), 30 (H784Y), 31 (H784W), and35 (H784K), and single bands corresponding to the desired recombinantprotein were visualized (data not shown).

The purified enzymes were tested for nuclease contamination usingDNaseAlert™ and RNaseAlert® nuclease detection kits (Integrated DNATechnologies, Coralville, Iowa) following protocols recommended by themanufacturer. All enzyme preparations were determined to be free ofcontaminating nucleases.

Example 4 Characterization of Properties of 18 Mutant Taq DNAPolymerases in PCR

The 18 mutant Taq DNA polymerase enzymes described in Example 3 werecharacterized for polymerase activity and the ability to discriminate a3′-RNA residue in the primer oligonucleotide.

The unit activity of the purified wild-type protein was determined bycomparing performance in qPCR of known quantities of OptiTaq and eachmutant compared to a commercial non-hot-start Taq DNA polymerase, Taq-BDNA Polymerase (Enzymatics, Beverly, Mass.). Quantification cycle values(Cq, the amplification cycle number at which positive signal is firstdetected) and amplification curve shapes were analyzed to determine thenanogram amounts at which both enzymes performed similarly in thesuboptimal range for each. Using these nanogram amounts and known unitvalues of Taq-B DNA polymerase, relative activity unit values could beextrapolated for all of the mutant DNA polymerase enzymes havingsufficient activity to support PCR.

The following reaction conditions were employed: 1× qPCR buffer (20 mMTris pH 8.4, 50 mM KCl, 3 mM MgCl₂, 0.01% Triton-X100), 800 μM dNTPs(200 μM each), 500 nM For primer (Hs HPRT F517, SEQ ID NO. 43), 500 nMRev primer (Hs HPRT R591, SEQ ID NO. 44), 250 nM probe (Hs HPRT P554,SEQ ID NO. 45), 2×10³ copies of linearized cloned plasmid template(HPRT-targ, SEQ ID NO. 46), in 10 μL final volume. The amount of DNApolymerase added to each reaction was varied as follows: for wild type(OptiTaq), reactions were set using 10, 1, 0.1, 0.01, and 0.001 U/μL(220, 22, 2.2, 0.22, or 0.022 ng of protein per 10 μL reaction). Mutantpolymerases were run in similar concentrations. In addition, thosemutant enzymes showing polymerase activity were more finely titratedtesting 220, 22, 10.6, 4.8, 2.2, 1.1, 0.48, and 0.22 ng of protein per10 μL reaction. Enzyme dilutions were made in enzyme dilution buffer (20mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1% Triton-X100, 1 mg/mL BSA,10% glycerol). Reactions were run in 384 well format on a BIO-RADCFX384™ Real-Time System (BIO-RAD, Hercules, Calif.) using cyclingparameters 95° C. for 30 seconds followed by 60 cycles of [95° C. for 15seconds followed by 60° C. for 1 minutes]. Detection was achieved usinga fluorescence-quenched probe (5′-nuclease assay format, note that themutations introduced into the present series of Taq mutants do not liein the 5′-nuclease domain). Sequences of the primers, probe, andtemplate (plasmid insert) are shown in Table 6.

TABLE 6 Sequence of oligonucleotides employed in Taq DNA polymeraseactivity assay. Name Sequence SEQ ID NO. Hs HPRT GACTTTGCTTTCCTTGGTCAGSEQ ID NO. 43 F517 Hs HPRT GGCTTATATCCAACACTTCGTG SEQ ID NO. 44 R591Hs HPRT FAM-ATGGTCAAG(ZEN)GTCGCAAGCTTGCTGGT-IBFQ SEQ ID NO. 45 P554HPRT- GACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAAAGATG SEQ ID NO. 46 targGTCAAGGTCGCAAGCTTGCTGGTGAAAAGGACCCCACGAA GTGTTGGATATAAGCC Nucleic acidsequences are shown 5′-3′. FAM = 6-carboxyfluorescein, IBFQ = Iowa BlackFQ (fluorescence quencher), and ZEN = ZEN internal fluorescencequencher.

These 18 Taq DNA polymerase mutants were characterized as outlinedabove. Results are summarized in Table 7. Six mutants, including MutantIDs 4, 5, 9 12, 13, and 17, did not show detectable DNA polymeraseactivity and were not studied further. Six mutants, Mutant IDs 6, 7, 11,14, 15, and 16 had DNA polymerase activity; however, processivity wasreduced from 4-50 fold relative to the wild type enzyme. Six mutants,Mutant IDs 1, 2, 3, 8, 10, and 18, showed DNA polymerase activitysimilar to wild type OptiTaq.

TABLE 7 Novel Taq DNA polymerase mutants selected for initial study.Amino acid ΔCq Delay in Mutant changes from Polymerase Relative primingfrom ID wild-type Taq Activity activity* an RNA base** 1 V783I Yes 1 0 2V783F Yes 1 1 3 H784Q Yes 1 1 4 R573H No — — 5 Q582K No — — 6 F667W Yes0.25 9 7 H639W Yes 0.02 20  8 L616M Yes 1 0 9 E615L, L616E No — — 10A661E, I665W, Yes 1   2.9 F667L 11 Q782I, H784F Yes 0.20 2 12 Q782I,V783L, No — — H784L 13 Q782S, V783F, No — — H784N 14 Q782P, V783L, Yes0.02   2.5 H784Q 15 Q754A Yes 0.2 >35  16 R659H Yes 0.1 >35  17 V783F,H784Q No — — 18 V783L, H784Q Yes 1 1 *Wild-type OptiTaq was set to “1”and the relative activity of each of the mutant polymerases wasnormalized to this amplification efficiency, with 1 as the maximum.**ΔCq = [Cq Mutant ID X] − [Cq OptiTaq] when qPCR reactions are runusing primers having a 3′-RNA residue.

The subset of these mutant Taq DNA polymerases which showed DNApolymerase activity were studied for their ability to discriminatebetween primers having a 3′-DNA versus a 3′-RNA residue relative to thewild type OptiTaq enzyme. Real-time PCR was performed as before,employing in the reactions the amount of each mutant DNA polymeraseequal to 0.5 units of wild-type OptiTaq per 10 μL reaction. Thefollowing reaction conditions were employed: 1× qPCR buffer (20 mM TrispH 8.4, 50 mM KCl, 3 mM MgCl₂, 0.01% Triton-X100), 800 μM dNTPs (200 μMeach), 500 nM For primer (Hs SFRS9 F569 rU, SEQ ID NO. 47), 500 nM Revprimer (Hs SFRS9 R712 rA, SEQ ID NO. 48), 250 nM probe (Hs SFRS9 P644,SEQ ID NO. 49), 2×10³ copies of linearized cloned plasmid template(SFRS9-targ, SEQ ID NO. 50), in 10 μL final volume. Reactions were runin 384 well format on a BIO-RAD CFX384™ Real-Time System (BIO-RAD,Hercules, Calif.) using cycling parameters 95° C. for 30 secondsfollowed by 60 cycles of [95° C. for 15 seconds followed by 60° C. for 1minutes]. Detection was achieved using a fluorescence-quenched probe(5′-nuclease assay format). Sequences of the primers, probe, andtemplate (plasmid insert) are shown in Table 8.

TABLE 8 Sequence of oligonucleotides employed in the primer 3′-RNAdiscrimination assay. Name Sequence SEQ ID NO. Hs SFRS9TGTGCAGAAGGATGGAGu SEQ ID NO. 47 F569 rU Hs SFRS9 CTGGTGCTTCTCTCAGGATaSEQ ID NO. 48 R712 rA Hs SFRS9 HEX-TGGAATATG(ZEN)CCCTGCGTAAACTGGA-IBFQSEQ ID NO. 48 P644 SFRS9- TGTGCAGAAGGATGGAGTGGGGATGGTCGAGTATCTCAGSEQ ID NO. 50 targ AAAAGAAGACATGGAATATGCCCTGCGTAAACTGGATGACACCAAATTCCGCTCTCATGAGGGTGAAACTTCCTACAT CCGAGTTTATCCTGAGAGAAGCACCAGNucleic acid sequences are shown 5′-3′ with DNA uppercase and RNAlowercase. HEX = hexachlorofluorescein, IBFQ = Iowa Black FQ(fluorescence quencher), and ZEN = ZEN fluorescence quencher.

The 12 Taq DNA polymerase mutants that supported PCR were tested for theability to use a 3′-RNA modified primer as outlined above. Results aresummarized in Table 7. Mutant IDs 1 and 8 did not show any differencebetween primers having a 3′-DNA versus a 3′-RNA residue. Mutant IDs 2,3, 6, 7, 10, 11, 14, 15, 16, and 18 showed an amplification delay using3′-RNA primers. Thus the rational design strategy employed herein wassuccessful and Taq DNA polymerase mutants were identified whichdiscriminated against priming from a 3′-RNA residue. Those mutants whichshowed some delay with RNA priming and showed high processivity werestudied for improvements in primer 3′-residue mismatch discrimination.

Example 5 Improved Mismatch Discrimination in Allele-Specific PCR UsingMutant Taq DNA Polymerases

Of the 18 mutant enzymes studied in Example 4, Mutant IDs 2, 3, 10, and18 showed the ability to discriminate against a 3′-RNA residue in theprimer and retained high enzymatic activity/processivity. These fourmutants were studied for the ability to discriminate against a3′-terminal DNA mismatch compared with wild type OptiTaq DNA polymeraseusing an allele-specific qPCR assay. Amplification reactions wereperformed against a synthetic oligonucleotide template where a singlebase was varied (SNP) which was positioned to lie at the 3′-end of thereverse primer. Synthetic templates were employed having each of the 4possible bases at this position. Reverse primers were employed havingeach of the 4 possible bases at the 3′-end. Relative amplificationefficiency for all pairwise combinations was assessed using qPCR.

Quantitative allele-specific real-time PCR (AS-qPCR) was performed in 10μL reaction volumes in 384 well format with 2×10³ copies of a 103 bpsynthetic template (SEQ ID NOs. 51-4). Final reaction conditions usedwere 20 mM Tris-HCL (pH 8.4 at 25° C.), 50 mM KCL, and 3 mM MgCl₂, 0.01%Triton X-100, 800 μM total dNTPs, and 200 nM of the universal forwardprimer (SEQ ID NO. 60), 200 nM of a reverse primer (separate reactionswere set up for each of the allele-specific primers SEQ ID NOs. 55-58 orthe control universal primer SEQ ID NO. 59) and 200 nM of the 5′nuclease detection probe (SEQ ID NO. 61). Each allele-specific primerwas tested on each SNP template. Reactions utilized either 0.5 U (10.8ng/11.1 nM/111 fmol) of the wild type OptiTaq DNA polymerase or 0.5 U ofone of the 4 Taq DNA polymerase mutants studied (MUT ID No. 2 V783F, MUTID NO. 3 H784Q, MUT ID NO. 10 A661E I665W F667L, or MUT ID NO. 18 V783LH784Q). Amplification was performed on a CFX384™ C1000™ Thermo Cyclersystem (Bio-Rad, Hercules, Calif.) using the following cyclingparameters: 95° C. for 30 seconds initial denaturation followed by 60cycles of 95° C. for 10 seconds, then 60° C. for 30 seconds.Oligonucleotide reagents used in this example are shown in Table 9.

TABLE 9 Synthetic oligonucleotides employed in Example 5. NameSequence (5′-3′) SEQ ID NO. A TemplateAGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAA SEQ ID NO. 51GTTGGCCTCAGAAGTAGTGGCCAGCTGTGTGTCGGGGAACAGT AAAGGCATGAAGCTCAG C TemplateAGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAA SEQ ID NO. 52GTTGGCCTCAGAAGTAGTGGCCAGCTGTGTGTCGGGGCACAGT AAAGGCATGAAGCTCAG G TemplateAGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAA SEQ ID NO. 53GTTGGCCTCAGAAGTAGTGGCCAGCTGTGTGTCGGGGGACAGT AAAGGCATGAAGCTCAG T TemplateAGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAA SEQ ID NO. 54GTTGGCCTCAGAAGTAGTGGCCAGCTGTGTGTCGGGGTACAGT AAAGGCATGAAGCTCAG Syn Rev TCTGAGCTTCATGCCTTTACTGTT SEQ ID NO. 55 Syn Rev C CTGAGCTTCATGCCTTTACTGTCSEQ ID NO. 56 Syn Rev A CTGAGCTTCATGCCTTTACTGTA SEQ ID NO. 57 Syn Rev GCTGAGCTTCATGCCTTTACTGTG SEQ ID NO. 58 Syn Rev CTGAGCTTCATGCCTTTACTGTSEQ ID NO. 59 Syn For AGCTCTGCCCAAAGATTACCCTG SEQ ID NO. 60 Syn ProbeFAM-TTCTGAGGC(ZEN)CAACTTCCACTGCCACTTA-IBFQ SEQ ID NO. 61 DNA bases areuppercase; FAM = 6-carboxyfluorescein; IBFQ = Iowa Black™ FQfluorescence quencher; ZEN = internal ZEN fluorescence quencher;underlined base indicates the SNP site in the synthetic template DNA.

Initially all reactions were run in triplicate. Similar results wereobtained for all replicates when using the wild type OptiTaq. However,results showed greater variation for the mutant polymerases. To obtainstatistically meaningful results, each reaction was therefore performed96 times for the mutant polymerases and 81 times for the wild typeenzyme. ΔCq values were calculated as the Cq value obtained for eachmismatched base pair minus the Cq value obtained for the matched basepair (ΔCq=Cq mismatch−Cq match). The ΔCq values for all 96 replicateswere averaged and standard deviations were calculated. Results are shownin Table 10 and are graphically summarized in FIGS. 3A and 3B. Note thatthe reverse primer is the allele-specific primer, so the “Syn Rev T”primer (SEQ ID NO. 55) is the perfect match to the Template A (SEQ IDNO. 51), etc.

TABLE 10 ΔCq values for AS-qPCR reactions using WT OptiTaq and mutantTaq DNA polymerases. Reverse Primer Template SEQ A C G T DNA ID SEQ IDSEQ ID SEQ ID SEQ ID Polymerase Name NO. NO. 51 NO. 52 NO. 53 NO. 54OptiTaq Syn Rev T 55 — 2.3 +/− 0.2  1.4 +/− 0.2 3.8 +/− 0.2 Syn Rev G 587.6 +/− 0.6 —  5.6 +/− 0.3 1.9 +/− 0.2 Syn Rev C 56 1.8 +/− 0.2 7.6 +/−0.6 — 2.0 +/− 0.2 Syn Rev A 57 6.6 +/− 0.4 1.5 +/− 0.2  8.0 +/− 0.6 —MUT ID 2 Syn Rev T 55 — 7.3 +/− 2.9  4.5 +/− 0.5 9.5 +/− 1.8 V783F SynRev G 58 17.9 +/− 8.3  — 16.4 +/− 7.5 4.1 +/− 0.2 Syn Rev C 56 6.5 +/−1.2 15.0 +/− 8.9  — 5.3 +/− 0.5 Syn Rev A 57 7.8 +/− 4.0 3.5 +/− 0.414.6 +/− 9.7 — MUT ID 3 Syn Rev T 55 — 7.5 +/− 0.8  7.0 +/− 0.6 10.4 +/−2.3  H784Q Syn Rev G 58 13.3 +/− 7.6  — 10.1 +/− 4.8 4.6 +/− 0.2 Syn RevC 56 6.9 +/− 0.5 8.6 +/− 2.6 — 5.6 +/− 0.4 Syn Rev A 57 17.1 +/− 7.2 6.3 +/− 0.5 21.2 +/− 8.7 — MUT ID 10 Syn Rev T 55 — 9.0 +/− 0.9  5.7 +/−0.3 11.2 +/− 2.6  A661E Syn Rev G 58 19.9 +/− 8.4  — 13.9 +/− 5.3 3.9+/− 0.3 I665W Syn Rev C 56 8.7 +/− 4.3 19.2 +/− 9.7  — 7.4 +/− 0.8 F667LSyn Rev A 57 13.3 +/− 8.2  6.1 +/− 0.8 13.1 +/− 8.6 — MUT ID 18 Syn RevT 55 — 5.8 +/− 1.3  6.0 +/− 0.4 9.4 +/− 1.2 V783L Syn Rev G 58 22.7 +/−8.0  — 18.9 +/− 8.4 4.9 +/− 0.3 H784Q Syn Rev C 56 6.8 +/− 0.5 17.6 +/−9.6  — 4.8 +/− 0.4 Syn Rev A 57 19.3 +/− 8.2  6.1 +/− 0.4 26.6 +/− 6.4 —Average ΔCq values are shown, where ΔCq = [Cq mismatch − Cq match], +/−standard deviation calculated from 96 replicates.

The wild type OptiTaq showed an average ΔCq for AS-qPCR in thissynthetic amplicon system of 4.2 with a range of 1.4 to 8.0. Mutant ID 2(V783F) showed an average ΔCq of 9.4 with a range of 3.5 to 17.9. MutantID 3 (H784Q) showed an average ΔCq of 9.9 with a range of 4.6 to 21.2.Mutant ID 10 (A661E, I665W, F667L) showed an average ΔCq of 10.9 with arange of 3.9 to 19.9. Mutant ID 18 (V783L, H784Q) showed an average ΔCqof 12.4 with a range of 4.9 to 26.6. Therefore in all pairwisecombinations of 4 template bases and 4 3′-terminal primer bases themutant Taq DNA polymerases of the present invention showed greaterdiscrimination to mismatch than did the wild type OptiTaq DNApolymerase. The magnitude of improvement for each mismatch pair isdefined by the ΔΔCq, which is the difference of discrimination betweenthe mutant and wild type enzymes (ΔΔCq=ΔCq mutant−ΔCq wild type). TheΔΔCq values were calculated and are shown in Table 11.

TABLE 11 ΔΔCq values for AS-qPCR reactions for the mutant Taq DNApolymerases compared with wild type OptiTaq. Reverse Primer Template SEQA C G T DNA ID SEQ ID SEQ ID SEQ ID SEQ ID Polymerase Name NO. NO. 51NO. 52 NO. 53 NO. 54 MUT ID Syn Rev T 55 — 5.0 3.1 5.7 NO. 2 Syn Rev G58 10.3  — 10.8  2.2 V783F Syn Rev C 56 4.7 7.4 — 3.3 Syn Rev A 57 1.22.0 6.6 — MUT ID Syn Rev T 55 — 5.2 5.6 6.6 NO. 3 Syn Rev G 58 5.7 — 4.52.7 H784Q Syn Rev C 56 5.1 1.0 — 3.6 Syn Rev A 57 10.5  4.8 13.2  — MUTID Syn Rev T 55 — 6.7 4.3 7.4 NO. 10 Syn Rev G 58 12.3  — 8.3 2.0 A661ESyn Rev C 56 6.9 11.6  — 5.4 I665W Syn Rev A 57 6.7 4.6 5.1 — F667L MUTID Syn Rev T 55 — 3.5 4.6 5.6 NO. 18 Syn Rev G 58 15.1  — 13.3  3.0V783L Syn Rev C 56 5.0 10.0  — 2.8 H784Q Syn Rev A 57 12.7  4.6 18.6  —Average ΔΔCq values are shown, where ΔΔCq = [ΔCq mutant − ΔCq wildtype], from data in Table 10.

Mutant ID 2 (V783F) showed an average ΔΔCq of 5.2 compared to wild typeOptiTaq. Mutant ID 3 (H784Q) showed an average ΔΔCq of 5.7 compared towild type OptiTaq. Mutant ID 10 (A661E, 1665W, F667L) showed an averageΔΔCq of 6.7 compared to wild type OptiTaq. Mutant ID 18 (V783L, H784Q)showed an average ΔΔCq of 8.2 compared to wild type OptiTaq. Thereforeeach of the mutant Taq DNA polymerases of the present invention showed asignificant improvement over wild type OptiTaq in mismatchdiscrimination, and, importantly, mismatch discrimination was improvedfor every possible mismatch base pair combination. Overall, mutant ID 18(V783L, H784Q) showed the best SNP discrimination within the set of 4mutant enzymes studied in this example using an AS-PCR assay.

Example 6 Discrimination Against a Primer 3′-RNA Residue by Taq DNAPolymerase Mutants

All 18 Taq DNA polymerase mutants were screened for the ability todiscriminate against priming from a 3′-RNA residue in Example 4. Thefour mutants studied in AS-PCR in Example 5 (MUT IDs 2, 3, 10, and 18)which showed good 3′-mismatch discrimination were studied in greaterdetail in the present example for the ability to discriminate againstthe presence of a 3′-terminal RNA residue in the primer, examining forpossible base-specific effects. Amplification reactions were performedagainst a synthetic oligonucleotide template where a single base wasvaried (SNP) which was positioned to lie at the 3′-end of the reverseprimer. Synthetic templates were employed having each of the 4 possiblebases at this position. Reverse primers were employed having each of the4 possible RNA bases at the 3′-end and results were compared to controlreactions using primers having each of the 4 possible DNA bases at the3′-end. Relative amplification efficiency was assessed using qPCR.

Quantitative real-time PCR (qPCR) was performed in 10 μL reactionvolumes in 384 well format with 2×10³ copies of a 103 bp synthetictemplate (SEQ ID NOs. 51-54). Final reaction conditions used were 20 mMTris-HCL (pH 8.4 at 25° C.), 50 mM KCL, and 3 mM MgCl₂, 0.01% TritonX-100, 800 μM total dNTPs, and 200 nM of the universal forward primer(SEQ ID NO. 60), 200 nM of a reverse primer (separate reactions were setup for each of the four 3′-RNA primers SEQ ID NOs. 62-65, each of thefour 3′DNA primers SEQ ID NOs. 55-58, or the control universal primerSEQ ID NO. 59) and 200 nM of the 5′ nuclease detection probe (SEQ ID NO.61). Each primer was tested only on the complementary template (mismatchconditions were not tested). Reactions utilized either 0.5 U (10.8ng/11.1 nM/111 fmol) of the wild type OptiTaq DNA polymerase or 0.5 U ofone of the 4 Taq DNA polymerase mutants studied (MUT ID No. 2 V783F, MUTID NO. 3 H784Q, MUT ID NO. 10 A661E I665W F667L, or MUT ID NO. 18 V783LH784Q). Amplification was performed on a CFX384™ C1000™ Thermo Cyclersystem (Bio-Rad, Hercules, Calif.) using the following cyclingparameters: 95° C. for 30 seconds initial denaturation followed by 60cycles of 95° C. for 10 seconds, then 60° C. for 30 seconds.Oligonucleotide reagents used in this example are shown in Table 12. Atotal of 96 replicates were performed for each pairwise combination.

TABLE 12 Synthetic oligonucleotides employed in Example 6. NameSequence (5′-3′) SEQ ID NO. A TemplateAGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAA SEQ ID NO. 51GTTGGCCTCAGAAGTAGTGGCCAGCTGTGTGTCGGGGAACAGT AAAGGCATGAAGCTCAG C TemplateAGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAA SEQ ID NO. 52GTTGGCCTCAGAAGTAGTGGCCAGCTGTGTGTCGGGGCACAGT AAAGGCATGAAGCTCAG G TemplateAGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAA SEQ ID NO. 53GTTGGCCTCAGAAGTAGTGGCCAGCTGTGTGTCGGGGGACAGT AAAGGCATGAAGCTCAG T TemplateAGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAA SEQ ID NO. 54GTTGGCCTCAGAAGTAGTGGCCAGCTGTGTGTCGGGGTACAGT AAAGGCATGAAGCTCAG Syn Rev TCTGAGCTTCATGCCTTTACTGTT SEQ ID NO. 55 Syn Rev C CTGAGCTTCATGCCTTTACTGTCSEQ ID NO. 56 Syn Rev A CTGAGCTTCATGCCTTTACTGTA SEQ ID NO. 57 Syn Rev GCTGAGCTTCATGCCTTTACTGTG SEQ ID NO. 58 Syn Rev rU CTGAGCTTCATGCCTTTACTGTuSEQ ID NO. 62 Syn Rev rC CTGAGCTTCATGCCTTTACTGTc SEQ ID NO. 63Syn Rev TA CTGAGCTTCATGCCTTTACTGTa SEQ ID NO. 64 Syn Rev rGCTGAGCTTCATGCCTTTACTGTg SEQ ID NO. 65 Syn Rev CTGAGCTTCATGCCTTTACTGTSEQ ID NO. 59 Syn For AGCTCTGCCCAAAGATTACCCTG SEQ ID NO. 60 Syn ProbeFAM-TTCTGAGGC(ZEN)CAACTTCCACTGCCACTTA-IBFQ SEQ ID NO. 61 DNA bases areuppercase and RNA bases are lowercase; FAM = 6-carboxyfluorescein; IBFQ= Iowa Black™ FQ fluorescence quencher; ZEN = internal ZEN fluorescencequencher; underlined base indicates the SNP site in the synthetictemplate DNA.

Average Cq values were calculated for the 96-replicate sets. ΔCq valueswere calculated as the difference between the average Cq values for the3′-RNA primer reactions from the average Cq values for the 3′-DNA primerreactions (ΔCq=Cq 3′-RNA−Cq 3′-DNA). Higher ΔCq values indicate agreater degree of discrimination against priming from a 3′-RNA primer.Results are shown in Table 13 and are graphically summarized in FIG. 4.

TABLE 13 ΔCq values for qPCR reactions using WT OptiTaq and mutant TaqDNA polymerases comparing 3′-DNA vs. 3′-RNA primers. DNA Reverse Primerscompared Polymerase Name SEQ ID NO. Template ΔCq OptiTaq Syn Rev T 55 A0.1 Syn Rev rU 62 SEQ ID NO. 51 Syn Rev G 58 C 0.2 Syn Rev rG 65 SEQ IDNO. 52 Syn Rev C 56 G 0.0 Syn Rev rC 63 SEQ ID NO. 53 Syn Rev A 57 T 0.1Syn Rev rA 64 SEQ ID NO. 54 MUT ID 2 Syn Rev T 55 A 5.4 V783F Syn Rev rU62 SEQ ID NO. 51 Syn Rev G 58 C 1.5 Syn Rev rG 65 SEQ ID NO. 52 Syn RevC 56 G 4.8 Syn Rev rC 63 SEQ ID NO. 53 Syn Rev A 57 T 2.2 Syn Rev rA 64SEQ ID NO. 54 MUT ID 3 Syn Rev T 55 A 6.9 H784Q Syn Rev rU 62 SEQ ID NO.51 Syn Rev G 58 C 2.0 Syn Rev rG 65 SEQ ID NO. 52 Syn Rev C 56 G 9.8 SynRev rC 63 SEQ ID NO. 53 Syn Rev A 57 T 1.4 Syn Rev rA 64 SEQ ID NO. 54MUT ID 10 Syn Rev T 55 A 5.5 A661E Syn Rev rU 62 SEQ ID NO. 51 I665W SynRev G 58 C 1.3 F667L Syn Rev rG 65 SEQ ID NO. 52 Syn Rev C 56 G 4.2 SynRev rC 63 SEQ ID NO. 53 Syn Rev A 57 T 0.8 Syn Rev rA 64 SEQ ID NO. 54MUT ID 18 Syn Rev T 55 A 9.3 V783L Syn Rev rU 62 SEQ ID NO. 51 H784Q SynRev G 58 C 2.4 Syn Rev rG 65 SEQ ID NO. 52 Syn Rev C 56 G 9.5 Syn Rev rC63 SEQ ID NO. 53 Syn Rev A 57 T 2.3 Syn Rev rA 64 SEQ ID NO. 54 AverageΔCq values are shown, where ΔCq = Cq 3′-RNA primer − Cq 3′-DNA primer.

Wild type OptiTaq did not show any significant discrimination between a3′-DNA and a 3′-RNA primer. All four mutant Taq DNA polymerases,however, showed reduced priming efficiency when using a 3′-RNA primer.Thus the goal of creating novel polymerases which discriminate against a3′-RNA residue in a primer was achieved using the intelligentmutagenesis design strategy described herein. Interestingly, themagnitude of discrimination was much greater for RNA pyrimidine residues(rC or rU) than for RNA purine residues (rA or rG).

Example 7 Improved Mismatch Discrimination in rhPCR using Mutant Taq DNAPolymerases

RNase H-based PCR (rhPCR) employs the enzyme RNase H2 to convert ablocked-cleavable oligonucleotide which cannot prime DNA synthesis intoa form that can prime DNA synthesis and initiate PCR. Theblocked-cleavable oligonucleotide, or blocked-cleavable primer, containsa single RNA residue near the 3′-end of the oligonucleotide (whichcomprises the cleavage site) and is modified at or near the 3′-end sothat the primer cannot prime DNA synthesis and/or has lost templatefunction and so is incompetent to support PCR even if primer extensioncan occur. This method can be used for genotyping (SNP discrimination)and relies on the ability of RNase H2 to distinguish between base-pairmatch vs. mismatch at the RNA base cleavage site when hybridized to thetarget nucleic acid. In rhPCR, SNP discrimination occurs at the primerunblocking step, not at the primer extension step (in AS-PCR,discrimination occurs at the primer extension step). Examples of thisenzyme cleaving strategy, similar RNase H strategies, and methods ofblocking primer extension or inhibiting template function and therebydisabling PCR are described in U.S. Pat. No. 7,112,406 to Behlke et al.,entitled POLYNOMIAL AMPLIFICATION OF NUCLEIC ACIDS, U.S. Pat. No.5,763,181 to Han et al., entitled CONTINOUS FLUOROMETRIC ASSAY FORDETECTING NUCLEIC ACID CLEAVAGE, U.S. Pat. No. 7,135,291 to Sagawa etal., entitled METHOD OF DETECTING NUCLEOTIDE POLYMORPHISM; U.S. Pat.App. No. 20090068643 to Behlke and Walder, entitled DUAL FUNCTIONPRIMERS FOR AMPLIFYING DNA AND METHODS OF USE; and U.S. Pat. App. No.20100167353 to Walder et al., entitled RNASE H-BASED ASSAYS UTILIZINGMODIFIED RNA MONOMERS and in Dobosy et al., RNase H-dependent PCR(rhPCR): improved specificity and single nucleotide polymorphismdetection using blocked cleavable primers, BMC Biotechnology., 11:e80(2011).

In AS-PCR the SNP is positioned at the 3′-end of the primer. In thisconfiguration, a mispriming event (where DNA synthesis is initiated inthe presence of a 3′-terminal mismatch) leads to incorporation of thebase present in the primer into the nascent DNA strand and thereby intothe PCR amplicon. This event converts the PCR product to the primersequence so that the amplified DNA now matches primer and no longermatches the original input sample nucleic acid sequence. Since theamplicon sequence now matches the primer and not the input sample,amplification proceeds at high efficiency.

In rhPCR, cleavage of the blocked-cleavable primer by RNase H2 occurs atthe 5′-side of the RNA residue; if the SNP is positioned at the RNAresidue (e.g., the RNA base pairs with the SNP), then the first baseincorporated by DNA polymerase during primer extension and PCR is theSNP site and results in daughter products which remain identical to theinput nucleic acid sequence. Rarely, non-canonical RNase H2 cleavageoccurs at the 3′-side of the RNA base, which leaves the RNA residue atthe 3′-end of the primer positioned overlying the SNP. In this case, therhPCR reaction proceeds like AS-PCR, where the 3′-end of the primer ispositioned at the SNP site and is either a match or mismatch to thetarget nucleic acid. Like AS-PCR, in the case of a mismatch, thesequence of the DNA extension product and PCR amplicon converts to thesequence of the primer and thus might not faithfully replicate thesequence of the sample during amplification. Any method which reducesthe frequency of this undesired mispriming event will improve mismatchdiscrimination in the rhPCR assay. Therefore, although basediscrimination in rhPCR is primarily mediated by RNase H2 at the primercleavage stage, use of a DNA polymerase that has an improved ability todiscriminate against a 3′-terminal mismatch and/or a 3′-terminal RNAresidue may improve the overall mismatch discrimination capacity ofrhPCR by preventing extension when undesired 3′-cleavage events occur.The DNA polymerase mutants described herein both reduce primingefficiency when a 3′-mismatch is present (improve mismatchdiscrimination) and reduce priming efficiency when a 3′-terminal RNAresidue is present in the primer (discriminate against a primer 3′-RNAresidue) compared with wild type Taq DNA polymerase. The present exampledemonstrates that the novel mutant Taq DNA polymerases of the presentinvention improve specificity and SNP discrimination of rhPCR.

Quantitative real-time rhPCR was performed comparing performance of wildtype OptiTaq DNA polymerase with mutant Taq DNA polymerases Mutant IDs2, 3, 10, and 18. Two different blocked-cleavable primer designs weretested, including the generation 1 (Gen1) “RDDDDx” primers and thegeneration 2 (Gen2) “RDxxD” primers (see: US Patent Application2012/0258455 by Behlke et al., entitled, RNASE H-BASED ASSAYS UTILIZINGMODIFIED RNA MONOMERS). Amplification reactions were performed using thesame synthetic oligonucleotide template employed in Example 5 where asingle base was varied (the SNP site) which was positioned to lie at theRNA residue in both Gen1 and Gen2 blocked-cleavable (rhPCR) primers.Synthetic templates were employed having each of the 4 possible bases atthis position. Reverse primers were employed having each of the 4possible complementary bases at this position (the RNA base). The sameforward primer was used for all reactions. Relative amplificationefficiency was assessed using real-time PCR.

Quantitative rhPCR was performed in 10 μL reaction volumes in 384 wellformat with 2×10⁶ copies of a 103 bp synthetic template (SEQ ID NOs.51-4). Final reaction conditions used were 20 mM Tris-HCL (pH 8.4 at 25°C.), 50 mM KCL, 3 mM MgCl₂, 0.01% Triton X-100, 800 μM total dNTPs, 200nM of the universal forward primer (SEQ ID NO. 60), 200 nM of a reverseprimer, and 200 nM of the 5′ nuclease detection probe (SEQ ID NO. 61).Reverse primers included Gen1 RDDDDx configuration allele-specific rhPCRprimers (SEQ ID NOs. 66-69), Gen2 RDxxD configuration allele-specificrhPCR primers (SEQ ID NOs. 70-73) and a control universal reverse primer(SEQ ID NO.59). Each of the rhPCR blocked-cleavable reverse primers weretested on each of the four SNP templates. Reactions utilized either 0.5U (10.8 ng/11.1 nM/111 fmol) of the wild type OptiTaq DNA polymerase or0.5 U of one of the four Taq DNA polymerase mutants (MUT ID 2, V783F;MUT ID 3, H784Q; MUT ID 10, A661E I665W F667L; or MUT ID 18, V783LH784Q). P. abyssi RNase H2 was added to each reaction in 1 μL volume.Reactions using the control and Gen1 blocked-cleavable RDDDDx rhPCRprimers employed 2.6 mU RNase H2 per 10 μL reaction (5 fmoles, 0.5 nMenzyme). Reactions using the Gen2 blocked-cleavable RDxxD rhPCR primersemployed 25 mU RNase H2 10 μL reaction (48 fmoles, 4.8 nM enzyme) forthe rC and rA primers (SEQ ID NOs. 71 and 72) and 200 mU RNase H2 per 10μL reaction (384 fmoles, 38 nM enzyme) for the rG and rU primers (SEQ IDNOs. 70 and 73). Cycling was performed on a Roche LightCycler® 480(Roche Applied Science, Indianapolis, Ind., USA) as follows: 95° C. for3 minutes followed by 75 cycles of 95° C. for 10 seconds and 60° C. for30 seconds. All reactions were performed in triplicate. Oligonucleotidereagents used in this example are shown in Table 14.

TABLE 14 Synthetic oligonucleotides employed in Example 7. NameSequence (5′-3′) SEQ ID NO. A TemplateAGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAA SEQ ID NO. 51GTTGGCCTCAGAAGTAGTGGCCAGCTGTGTGTCGGGGAACAGT AAAGGCATGAAGCTCAG C TemplateAGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAA SEQ ID NO. 52GTTGGCCTCAGAAGTAGTGGCCAGCTGTGTGTCGGGGCACAGT AAAGGCATGAAGCTCAG G TemplateAGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAA SEQ ID NO. 53GTTGGCCTCAGAAGTAGTGGCCAGCTGTGTGTCGGGGGACAGT AAAGGCATGAAGCTCAG T TemplateAGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAA SEQ ID NO. 54GTTGGCCTCAGAAGTAGTGGCCAGCTGTGTGTCGGGGTACAGT AAAGGCATGAAGCTCAG Syn RevCTGAGCTTCATGCCTTTACTGT SEQ ID NO. 59 Syn For AGCTCTGCCCAAAGATTACCCTGSEQ ID NO. 60 Syn Probe FAM-TTCTGAGGC(ZEN)CAACTTCCACTGCCACTTA-IBFQSEQ ID NO. 61 Syn Rev rU CTGAGCTTCATGCCTTTACTGTuCCCCx SEQ ID NO. 66DDDDx Syn Rev rC CTGAGCTTCATGCCTTTACTGTcCCCCx SEQ ID NO. 67 DDDDxSyn Rev rA CTGAGCTTCATGCCTTTACTGTaCCCCx SEQ ID NO. 68 DDDDx Syn Rev rGCTGAGCTTCATGCCTTTACTGTgCCCCx SEQ ID NO. 69 DDDDx Syn Rev rUCTGAGCTTCATGCCTTTACTGTuCxxC SEQ ID NO. 70 DxxD Syn Rev rCCTGAGCTTCATGCCTTTACTGTcCxxC SEQ ID NO. 71 DxxD Syn Rev rACTGAGCTTCATGCCTTTACTGTaCxxC SEQ ID NO. 72 DxxD Syn Rev rGCTGAGCTTCATGCCTTTACTGTgCxxC SEQ ID NO. 73 DxxD DNA bases are uppercaseand RNA bases are lowercase; FAM = 6-carboxyfluorescein; IBFQ = IowaBlack™ FQ fluorescence quencher; ZEN = internal ZEN fluorescencequencher; underlined base indicates the SNP site in the synthetictemplate DNA; “x” = C3 Spacer (propanediol).

MUT ID 10 (A661E, 1665W, F667L) unexpectedly showed large amplificationdelays when the primers matched the SNP site in the target in the rhPCRreactions using this synthetic amplicon system. This polymerase,however, did not show any delays when using a human genomic DNA systemfor rhPCR (see Examples 8 and 9). MUT ID 10 was therefore excluded fromanalysis in the synthetic system experiments. Data generated using theother three mutant polymerases were analyzed and ΔCq values werecalculated comparing matched versus mismatched primer/template pairs,where ΔCq=Cq mismatch−Cq match. Results are shown in Table 15 for theGen1 RDDDDx blocked-cleavable rhPCR primers and in Table 16 for the Gen2RDxxD blocked-cleavable rhPCR primers.

TABLE 15 ΔCq values for rhPCR reactions using WT OptiTaq and mutant TaqDNA polymerases with Gen1 RDDDDx blocked-cleavable rhPCR primers.Reverse Primer Template SEQ A G T C DNA ID SEQ ID SEQ ID SEQ ID SEQ IDPolymerase Name NO. NO. 51 NO. 53 NO. 54 NO. 52 OptiTaq Syn Rev 66 —10.5 3.4 6.6 rU DDDDx Syn Rev 67 3.3 — 1.3 2.2 rC DDDDx Syn Rev 68 9.510.5 — 3.5 rA DDDDx Syn Rev 69 9.5 10.8 11.8  — rG DDDDx MUT ID 2 SynRev 66 — 11.1 5.1 9.0 V783F rU DDDDx Syn Rev 67 4.0 — 1.9 3.6 rC DDDDxSyn Rev 68 10.4  11.1 — 5.6 rA DDDDx Syn Rev 69 10.2  10.5 10.7  — rGDDDDx MUT ID 3 Syn Rev 66 — 11.3 5.0 10.0  H784Q rU DDDDx Syn Rev 67 7.6— 4.3 5.9 rC DDDDx Syn Rev 68 10.8  11.3 — 7.6 rA DDDDx Syn Rev 69 10.9 10.9 11.0  — rG DDDDx MUT ID 18 Syn Rev 66 — 12.3 6.7 11.5  V783L rUDDDDx H784Q Syn Rev 67 9.9 — 8.3 10.4  rC DDDDx Syn Rev 68 11.5  13.2 —6.8 rA DDDDx Syn Rev 69 11.3  12.0 12.6  — rG DDDDx Average ΔCq valuesare shown, where ΔCq = [Cq mismatch − Cq match].

TABLE 16 ΔCq values for rhPCR reactions using WT OptiTaq and mutant TaqDNA polymerases with Gen2 RDxxD blocked-cleavable rhPCR primers. ReversePrimer Template SEQ A G T C DNA ID SEQ ID SEQ ID SEQ ID SEQ IDPolymerase Name NO. NO. 51 NO. 53 NO. 54 NO. 52 OptiTaq Syn Rev 70 —11.6 15.1 12.8 rU DxxD Syn Rev 71  6.3 —  6.7  4.6 rC DxxD Syn Rev 7213.7 15.6 — 14.3 rA DxxD Syn Rev 73 13.2 11.4 10.2 — rG DxxD MUT ID 2Syn Rev 70 — 12.2 15.0 14.0 V783F rU DxxD Syn Rev 71  8.3 —  6.5  4.4 rCDxxD Syn Rev 72 14.1 15.9 — 14.2 rA DxxD Syn Rev 73 13.8 12.2 11.6 — rGDxxD MUT ID 3 Syn Rev 70 — 12.4 15.0 14.1 H784Q rU DxxD Syn Rev 71  9.5—  7.8  6.4 rC DxxD Syn Rev 72 16.9 19.1 — 18.4 rA DxxD Syn Rev 73 15.013.0 12.7 — rG DxxD MUT ID 18 Syn Rev 70 — 13.0 15.3 14.3 V783L rU DxxDH784Q Syn Rev 71  6.9 —  9.6  3.6 rC DxxD Syn Rev 72 15.8 15.3 — 14.5 rADxxD Syn Rev 73 15.0 13.4 13.7 — rG DxxD Average ΔCq values are shown,where ΔCq = [Cq mismatch − Cq match].

In almost all cases, mismatch discrimination was superior for rhPCRreactions run using the mutant Taq DNA polymerases than wild typeOptiTaq. The magnitude of improvement is best seen by examining the ΔΔCqvalues, which is the difference of discrimination seen using wild typeOptiTaq and the mutants (ΔΔCq=ΔCq mutant−ΔCq wild type). These resultsare shown in Table 17 for the Gen1 RDDDDx primers and in Table 18 forthe Gen2 RDxxD primers. When using the Gen1 RDDDDx primers, the overallgreatest benefit was seen when the mismatched base was a “C” in thetarget nucleic acid and least benefit was seen when theblocked-cleavable primer contained a rG paired with a mismatched T inthe target. The greatest improvements were obtained using the mutant TaqDNA polymerase MUT ID 18 (V783L H784Q). The average ΔΔCq for MUT ID 2(V783F) was 1.0. The average ΔΔCq for MUT ID 3 (H784Q) was 2.0. Theaverage ΔΔCq for MUT ID 18 (V783L, H784Q) was 3.6. Benefits obtainedusing mutant Taq DNA polymerases was lower for the Gen2 RDxxD primers,which already showed high ΔCq values using wild type OptiTaq. AverageΔΔCq for the three mutant polymerases studied in the Example were 0.6,2.1, and 1.2. Therefore greatest benefit when using the Gen2 RDxxDprimers was seen with MUT ID 3 (H784Q).

TABLE 17 ΔΔCq values for rhPCR reactions using mutant Taq DNApolymerases compared with wild type OptiTaq for Gen1 RDDDDxblocked-cleavable rhPCR primers. Reverse Primer Template SEQ A G T C DNAID SEQ ID SEQ ID SEQ ID SEQ ID Polymerase Name NO. NO. 51 NO. 53 NO. 54NO. 52 MUT ID 2 Syn Rev 66 — 0.6 1.7 2.4 V783F rU DDDDx Syn Rev 67 0.7 —0.6 1.4 rC DDDDx Syn Rev 68 0.9 0.6 — 2.1 rA DDDDx Syn Rev 69 0.7 −0.3 1.1 — rG DDDDx MUT ID 3 Syn Rev 66 — 0.8 1.6 3.4 H784Q rU DDDDx Syn Rev67 4.3 — 3.0 3.7 rC DDDDx Syn Rev 68 1.3 0.8 — 4.1 rA DDDDx Syn Rev 691.4 0.1 −0.8  — rG DDDDx MUT ID 18 Syn Rev 66 — 1.8 3.3 4.9 V783L rUDDDDx H784Q Syn Rev 67 6.6 — 7.0 8.2 rC DDDDx Syn Rev 68 2.0 2.7 — 3.3rA DDDDx Syn Rev 69 1.8 1.2 0.8 — rG DDDDx ΔΔCq values are shown, whereΔΔCq = [ΔCq mutant − ΔCq wild type polymerase].

TABLE 18 ΔΔCq values for rhPCR reactions using mutant Taq DNApolymerases compared with wild type OptiTaq for Gen2 RDxxDblocked-cleavable rhPCR primers. Reverse Primer Template SEQ A G T C DNAID SEQ ID SEQ ID SEQ ID SEQ ID Polymerase Name NO. NO. 51 NO. 53 NO. 54NO. 52 MUT ID 2 Syn Rev 70 — 0.6 −0.1  1.2 V783F rU DxxD Syn Rev 71 2.0— −0.2  −0.2  rC DxxD Syn Rev 72 0.4 0.3 — −0.1  rA DxxD Syn Rev 73 0.60.8 1.4 — rG DxxD MUT ID 3 Syn Rev 70 — 0.8 −0.1  1.3 H784Q rU DxxD SynRev 71 3.2 — 1.1 1.8 rC DxxD Syn Rev 72 3.2 3.5 — 4.1 rA DxxD Syn Rev 732.8 1.6 2.5 — rG DxxD MUT ID 18 Syn Rev 70 — 1.4 0.2 1.5 V783L rU DxxDH784Q Syn Rev 71 0.6 — 2.9 −1.0  rC DxxD Syn Rev 72 2.1 −0.3  — 0.2 rADxxD Syn Rev 73 1.8 2.0 3.5 — rG DxxD ΔΔCq values are shown, where ΔΔCq= [ΔCq mutant − ΔCq wild type polymerase].

Example 8 Improved Mismatch Discrimination in rhPCR using Mutant Taq DNAPolymerases in a Human Genomic DNA SNP Assay

Example 7 demonstrated utility of the novel mutant Taq DNA polymerasesof the present invention in a synthetic amplicon rhPCR SNPdiscrimination assay system. The present Example demonstrates utility ofthe novel mutant Taq DNA polymerases in a human genomic DNA rhPCR SNPdiscrimination assays system, examining a SNP site in the SMAD7 gene(NM_005904, C/T SNP, rs4939827). The assays employed target DNAs GM18562(homozygous C/C) and GM18537 (homozygous T/T) from the Coriell Institutefor Medical Research (Camden, N.J., USA). Two differentblocked-cleavable primer designs were tested, including the generation 1(Gen1) “RDDDDx” primers and the generation 2 (Gen2) “RDxxD” primers(see: US Patent Application 2012/0258455 by Behlke et al., entitled,RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS).

Quantitative real-time rhPCR was performed in 10 μL reaction volumes in384 well format with 20 ng (the equivalent of 6600 copies of target) ofhuman genomic DNA (GM18562 or GM18537). Reactions utilized either 0.5 U(10.8 ng/11.1 nM/111 fmol) of wild type OptiTaq DNA polymerase or 0.5 Uof one of the four Taq DNA polymerase mutants (MUT ID 2, V783F; MUT ID3, H784Q; MUT ID 10, A661E I665W F667L; or MUT ID 18, V783L H784Q).Final reaction conditions used were 20 mM Tris-HCL (pH 8.4 at 25° C.),50 mM KCL, 3 mM MgCl₂, 0.01% Triton X-100, 800 μM total dNTPs, 200 nM ofa forward primer (SEQ ID NOs. 75-79), 200 nM of the universal reverseprimer (SEQ ID NO. 74), and 200 nM of the SMAD7 probe (SEQ ID NO. 80).Sequence of the 85 bp SMAD7 amplicon is shown as SEQ ID NO. 81. Forwardprimers included RDDDDx configuration Gen1 allele-specific rhPCR primers(SEQ ID NOs. 76 and 77), RDxxD configuration Gen2 allele-specific rhPCRprimers (SEQ ID NOs. 78 and 79) and the control universal forward primer(SEQ ID NO.75) which is not allele specific. Oligonucleotide reagentsemployed in this Example are shown in Table 19. Reactions included 1 μLof P.a. RNase H2 at a concentration of 2.6 mU per 10 μL reaction (5fmoles, 0.5 nM) for the Gen1 RDDDDx primers and control primer (SEQ IDNOs. 75-77) or 200 mU per 10 μL reaction (384 fmoles, 38.4 nM) for theGen2 RDxxD primers (SEQ ID NOs. 78 and 79). Amplification was performedon a Roche LightCycler® 480 (Roche Applied Science, Indianapolis, Ind.,USA) as follows: 95° C. for 3 minutes followed by 75 cycles of 95° C.for 10 seconds and 60° C. for 30 seconds. All reactions were performedin triplicate.

TABLE 19 Synthetic oligonucleotides employed in Example 8. SEQ ID NameSequence (5′-3′) NO. SMAD7 Rev CTCACTCTAAACCCCAGCATT 74 SMAD7 ForCAGCCTCATCCAAAAGAGGAAA 75 SMAD7 For rC CAGCCTCATCCAAAAGAGGAAAcAGGAx 76DDDDx SMAD7 For rU CAGCCTCATCCAAAAGAGGAAAuAGGAx 77 DDDDx SMAD7 For rCCAGCCTCATCCAAAAGAGGAAAcAxxA 78 DxxD SMAD7 For rUCAGCCTCATCCAAAAGAGGAAAuAxxA 79 DxxD SMAD7 probeFAM-CCCAGAGCTCCCTCAGACTCCT-IBFQ 80 SMAD7 targetCAGCCTCATCCAAAAGAGGAAATAGGACCCC 81 AGAGCTCCCTCAGACTCCTCAGGAAACACAGACAATGCTGGGGTTTAGAGTGAG DNA bases are uppercase and RNA bases arelowercase; FAM = 6-carboxyfluorescein; IBFQ = Iowa Black™ FQfluorescence quencher; “x” = C3 Spacer (propanediol). Primer and probebinding sites in the SMAD7 target are underlined.

Results using the Gen1 RDDDDx rhPCR primers are shown in Table 20 andusing the Gen2 RDxxD rhPCR primers are shown in Table 21. Overall, useof the mutant Taq DNA polymerases showed small but real improvements inSNP discrimination in this human genomic DNA rhPCR assay using the Gen1RDDDDx primers. However, large improvements in discrimination were seenusing the Gen2 RDxxD primers. The Gen2 RDxxD primers inherently showgreater SNP discrimination and these levels were increased so that ΔCqvalues are in some cases were greater than 40 amplification cyclesbetween match and mismatch; this level of discrimination would be“greater than assay” for most users, as qPCR reactions are seldom runfor over 45-50 cycles and positive signal was not detected in thesecases until after 70 cycles (Table 21). Therefore use of the new mutantTaq DNA polymerases improves SNP discrimination in rhPCR genotypingassays.

TABLE 20 SNP discrimination of a site in the SMAD7 gene using Gen1RDDDDx primers comparing wild type OptiTaq with four mutant Taq DNApolymerases. Cq Cq SEQ mU RNase Value Value DNA ID H2 per C/C T/TPolymerase For Primer NO. 10 μL rxn DNA DNA ΔCq Wild type SMAD7 For 752.6 24.3 25.3 — OptiTaq SMAD7 For 76 2.6 26.1 38.1 11.9 rC DDDDx SMAD7For 77 2.6 36.6 26.8  9.8 rU DDDDx MUT ID 2 SMAD7 For 75 2.6 24.7 25.5 —V783F SMAD7 For 76 2.6 26.2 40.3 14.1 rC DDDDx SMAD7 For 77 2.6 37.827.6 10.1 rU DDDDx MUT ID 3 SMAD7 For 75 2.6 25.3 27.1 — H784Q SMAD7 For76 2.6 26.2 46.1 19.9 rC DDDDx SMAD7 For 77 2.6 38.9 32.4  6.5 rU DDDDxMUT ID 10 SMAD7 For 75 2.6 24.3 25.8 — A661E SMAD7 For 76 2.6 25.6 43.918.3 I665W rC DDDDx F667L SMAD7 For 77 2.6 42.6 28.5 14.1 rU DDDDx MUTID 18 SMAD7 For 75 50 24.6 25.6 — V783L SMAD7 For 76 50 25.2 35.7 10.5H784Q rC DDDDx SMAD7 For 77 50 37.9 26.4 11.5 rU DDDDx DNA targetsincluded GM18562 (homozygous C/C) and GM18537 (homozygous T/T) from theCoriell Institute for Medical Research. ΔCq = [Cq mismatch − Cq match].

TABLE 21 SNP discrimination of a site in the SMAD7 gene using Gen2 RDxxDprimers comparing wild type OptiTaq with four mutant Taq DNApolymerases. Cq Cq SEQ mU RNase Value Value DNA ID H2 per C/C T/TPolymerase For Primer NO. 10 μL rxn DNA DNA ΔCq Wild type SMAD7 For 752.6 24.3 25.3 — OptiTaq SMAD7 For 78 200 25.9 40.4 14.5 rC DxxD SMAD7For 79 200 47.9 26.6 21.3 rU DxxD MUT ID 2 SMAD7 For 75 2.6 24.7 25.5 —V783F SMAD7 For 78 200 26.6 64.4 37.7 rC DxxD SMAD7 For 79 200 59.7 28.031.6 rU DxxD MUT ID 3 SMAD7 For 75 2.6 25.3 27.1 — H784Q SMAD7 For 78200 26.7 71.7 45.0 rC DxxD SMAD7 For 79 200 62.5 28.9 33.7 rU DxxD MUTID 10 SMAD7 For 75 2.6 24.3 25.8 — A661E SMAD7 For 78 200 25.6 74.4 48.8I665W rC DxxD F667L SMAD7 For 79 200 54.3 28.2 26.0 rU DxxD MUT ID 18SMAD7 For 75 50 24.6 25.6 — V783L SMAD7 For 78 200 25.1 52.7 27.6 H784QrC DxxD SMAD7 For 79 200 43.0 27.6 15.3 rU DxxD DNA targets includedGM18562 (homozygous C/C) and GM18537 (homozygous T/T) from the CoriellInstitute for Medical Research. ΔCq = [Cq mismatch − Cq match].

The ΔCq values for the SMAD7 SNP genotyping assays are graphicallysummarized in FIG. 5A for the Gen1 RDDDDx primers and in FIG. 6A for theGen2 RDxxD primers. It is interesting to note that, for the rhPCRgenotyping assays studied in Example 8, MUT ID 10 (A661E I665W F667L)showed the greatest improvement compare with wild type OptiTaq,especially when using the Gen2 RDxxD primers. Example 5 demonstratedutility of the mutant Taq DNA polymerases in AS-PCR, and in this caseuse of MUT ID 18 (V783L H784Q) showed the greatest benefit and MUT ID 3(H784Q) showed the next greatest relative benefit. It is clear that notonly do the different mutant Taq DNA polymerases of the presentinvention have utility in different amplification assays but that thedifferent mutants show varying levels of benefit depending on the natureof the assay used. It is therefore useful to have a collection of mutantpolymerases whose properties can be matched to differentassays/applications so that maximal benefit is obtained.

Example 9 Improved Discrimination of Rare Alleles in Genomic DNA usingrhPCR with Mutant Taq DNA Polymerases

Use of the Gen2 RDxxD blocked-cleavable primers in rhPCR can detect thepresence of a SNP at a level of 1:1,000 to 1:10,000 in the background ofwild type genomic DNA using native (wild type) Taq DNA polymerase (see:US Patent Application 2012/0258455 by Behlke et al., entitled, RNASEH-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS). The present exampledemonstrates that the mutant Taq DNA polymerases of the presentinvention improve rare allele discrimination in the rhPCR assay.

Rare allele detection experiments were designed to detect the baseidentity of a SNP site in the SMAD7 gene (NM_005904, C/T SNP, rs4939827)and employed target DNAs GM18562 (homozygous C/C) and GM18537(homozygous T/T) (Coriell Institute for Medical Research, Camden, N.J.,USA). Control reactions were set up using 2 ng (660 copies), 0.2 ng (66copies), or 0.02 ng (6.6 copies) of input matched target DNA. Rareallele detection reactions were set up using 2 ng (660 copies), 0.2 ng(66 copies), or 0.02 ng (6.6 copies) of input matched target DNA of oneallele plus 200 ng (66,000 copies) of the other (mismatched) allele.Background was established in reactions that contained 0 copies ofmatched target DNA plus 200 ng (66,000 copies) of the mismatched targetDNA. Both combinations were tested: GM18562 (C/C) as the rare allele inthe presence of excess GM18537 (T/T) and GM18537 (T/T) as the rareallele in the presence of excess GM18562 (C/C).

Quantitative real-time rhPCR was performed in 10 μL reaction volumes in384 well format. Final reaction conditions used were 10 mM Tris-HCL (pH8.4 at 25° C.), 50 mM KCL, 3.5 mM MgCl₂, 0.01% Triton-X100, 0.8 mMdNTPs, 200 nM of one of the SMAD7 forward primers (SEQ ID NOs. 75, 78,and 79), 200 nM of the SMAD7 reverse primer (SEQ ID NO. 74), and 200 nMof the SMAD7 probe (SEQ ID NO. 80). The 85 bp SMAD7 amplicon defined bythese primers is shown as SEQ ID NO. 81. Note that the forward primerswere either unmodified (control, SEQ ID NO. 75) or were specific for theSMAD7 C-allele (SEQ ID NO. 78) or the SMAD7 T-allele (SEQ ID NO. 79)using blocked-cleavable rhPCR Gen2 RDxxD design. Reactions utilizedeither 0.5 U of the wild type OptiTaq DNA polymerase or 0.5 U of one ofthe four Taq DNA polymerase mutants studied (MUT ID No. 2, V783F; MUT IDNO. 3, H784Q; MUT ID NO. 10, A661E I665W F667L; or MUT ID NO. 18, V783LH784Q). Reactions included P. abyssi RNase H2 at a concentration of 200mU per 10 μL reaction (384 fmoles) when using the SMAD7 For rC DxxD (SEQID NO. 78) primer and control reactions or 500-600 mU per 10 μL reaction(960-1152 fmoles) when using the SMAD7 For rU DxxD (SEQ ID NO. 79)primer. Oligonucleotide reagents used in this Example are shown in Table22. Cycling was performed on a Roche LightCycler® 480 (Roche AppliedScience, Indianapolis, Ind., USA) as follows: 95° C. for 3 minutesfollowed by 65 cycles of 95° C. for 10 seconds and 60° C. for 30seconds. All reactions were performed in triplicate.

TABLE 22 Synthetic oligonucleotides employed in Example 9. SEQ ID NameSequence (5′-3′) NO. SMAD7 Rev CTCACTCTAAACCCCAGCATT 74 SMAD7 ForCAGCCTCATCCAAAAGAGGAAA 75 SMAD7 For rC CAGCCTCATCCAAAAGAGGAAAcAxxA 78DxxD SMAD7 For rU CAGCCTCATCCAAAAGAGGAAAuAxxA 79 DxxD SMAD7 probeFAM-CCCAGAGCTCCCTCAGACTCCT-IBFQ 80 SMAD7 targetCAGCCTCATCCAAAAGAGGAAATAGGACCCC 81 AGAGCTCCCTCAGACTCCTCAGGAAACACAGACAATGCTGGGGTTTAGAGTGAG DNA bases are uppercase and RNA bases arelowercase; FAM = 6-carboxyfluorescein; IBFQ = Iowa Black™ FQfluorescence quencher; “x” = C3 Spacer (propanediol). Primer and probebinding sites in the SMAD7 target are underlined.

Results were analyzed and are shown in Table 23. The control columnsshow Cq values for matched primer/target reactions with no mismatchedtarget present and establish a quantification standard curve. The rareallele detection columns show Cq values for detection of 660, 66, 6, or0 (background control) copies of matched primer/target in the presenceof 66,000 copies of mismatched target. It is generally assumed that atleast a 3 cycle difference (ΔCq=3.0 or greater) between background andpositive signal is needed to call a reaction “positive” for rare alleledetection; a 5 cycle difference (ΔCq=5.0 or greater) is preferred. Inthis system, background is the signal observed when amplification isdone using no input target that is matched to the primer, so signalarises solely from amplification originating off the mismatched target.

Using wild type OptiTaq DNA polymerase, detection of the “C” allele inan excess of “T” background and detection of the “T” allele in an excessof “C” background both met the ΔCq 3.0 and ΔCq 5.0 levels of stringencyto call a 1:1000 rare allele detection event (66 copies of match targetin the presence of 66,000 copies of mismatch target). The 1:10,000reactions (6 copies of match target in the presence of 66,000 copies ofmismatch target) did not meet either of these criteria. Thus rhPCR had a1:1000 rare allele detection limit using wild type OptiTaq in thisgenomic DNA SNP system.

In contrast, rhPCR using each of the four mutants showed a 1:10,000 rareallele detection limit for both the “C” and “T” allele targets with aΔCq stringency cutoff of 3.0. MUT ID 3 (H784Q) showed a 1:10,000 rareallele detection limit for both the “C” and “T” targets in this genomicSNP system for the higher ΔCq stringency cutoff of 5.0. The other threemutant Taq DNA polymerases (MUT ID No. 2, V783F; MUT ID NO. 10, A661EI665W F667L; and MUT ID NO. 18, V783L H784Q) showed a 1:10,000 rareallele detection limit for the “C” allele target with a ΔCq stringencycutoff of 5.0 and a 1:10,000 rare allele detection limit for the “T”allele target with a ΔCq stringency cutoff of 3.0. We therefore concludethat the new mutant Taq DNA polymerases of the present invention providefor improved rare allele detection reactions using blocked-cleavableprimers in rhPCR compared with use of the wild type DNA polymerase.

TABLE 23 Rare allele detection using Gen2 RDxxD rhPCR primers comparingwild type OptiTaq with new mutant Taq DNA polymerases 200 ng mismatchedtemplate RNase H2 (66,000 copies of “wild type”) Control DNA SEQ ID per10 660 Match 66 Match 6 Match 0 Match (No mismatched template)Polymerase For Primer NO. μL rxn (1:100) (1:1,000) (1:10,000)(background) 660 Match 66 Match 6 Match 0 Match Wild type SMAD7 For 75200 mU 22.1 21.2 21.2 21.8 27.9 31.3 34.4 >65 OptiTaq SMAD7 For 78 200mU 28.2 31.5 35.1 37.0 28.8 33.3 37.3 >65 rC DxxD SMAD7 For 79 500 mU31.0 34.7 37.7 39.7 31.2 34.6 41.0 >65 rU DxxD MUT ID 2 SMAD7 For 75 200mU 22.2 22.2 22.1 22.2 28.9 32.7 35.7 >65 (V783F) SMAD7 For 78 200 mU28.2 31.7 35.4 45.4 29.0 33.3 37.5 >65 rC DxxD SMAD7 For 79 500 mU 28.632.5 36.7 41.3 28.2 34.0 42.0 >65 rU DxxD MUT ID 3 SMAD7 For 75 200 mU23.5 23.6 24.5 24.1 30.5 33.4 38.0 >65 (H784Q) SMAD7 For 78 200 mU 29.833.8 37.6 >65 30.5 35.5 39.6 >65 rC DxxD SMAD7 For 79 500 mU 32.9 37.744.0 52.3 30.1 35.9 44.9 >65 rU DxxD MUT ID 10 SMAD7 For 75 200 mU 22.222.4 22.5 22.8 28.3 31.9 35.5 >65 (A661E SMAD7 For 78 200 mU 31.8 34.738.5 59.3 30.0 33.9 37.8 >65 I665W rC DxxD F667L) SMAD7 For 79 600 mU33.5 38.4 43.2 46.2 31.9 36.5 41.0 >65 rU DxxD MUT ID 18 SMAD7 For 75200 mU 22.4 22.4 22.7 22.5 27.8 31.5 34.8 >65 (V783L SMAD7 For 78 200 mU28.8 32.9 37.5 46.5 29.5 33.4 37.8 >65 H784Q) rC DxxD SMAD7 For 79 500mU 30.1 34.0 38.4 41.8 29.4 36.0 44.7 >65 rU DxxD Cq values are shown.For the rare allele detection series (selective detection of 6-660 copesone genotype in the presence of 66,000 copies of the other genotype),those reactions having a ΔCq of 3.0 or better are highlighted in boldfont and those having a ΔCq of 5.0 or better are highlighted in boldfont with underline. ΔCq = [(Cq 0 copies match) − (Cq 6 copies match)],or ΔCq = [(Cq 0 copies match) − (Cq 66 copies match)], or ΔCq = [(Cq 0copies match) − (Cq 660 copies match)].

Example 10 Sequence of Taq DNA Polymerase Mutants Showing ImprovedDiscrimination for Mismatch or the Presence of an RNA Residue at the3′-End of the Primer

The complete amino acid and nucleotide sequences of the codon optimizedmutant enzymes employed in Examples 5-9 are shown below. Although thesesequences are easily derived from information provided in Tables 1, 3, 4and 5 by one with skill in the art, the final assembled sequences areprovided below for clarity. Base changes are identified in boldunderlined font for the nucleic acid and amino acid substitutions.

SEQ ID NO. 82, nucleotide sequence of Mutant ID 2 (V783F)CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAG TTCCATGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC.SEQ ID NO. 83, amino acid sequence of Mutant ID 2 (V783F)MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQ F HDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA.SEQ ID NO. 84, nucleotide sequence of Mutant ID 3 (H784Q)CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGGTC CAGGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC.SEQ ID NO. 85, amino acid sequence of Mutant ID3 (H784Q)MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQV Q DELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA.SEQ ID NO. 86, nucleotide sequence of Mutant ID 10 (A661E, I665W, F667L)CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGT GAA GCTAAAACATGG AAT T TGGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGGTCCATGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC.SEQ ID NO. 87, amino acid sequence of Mutant ID 10 (A661E, I665W, F667L)MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRR E AKT W N LGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA.SEQ ID NO. 88, nucleotide sequence of Mutant ID 18 (V783L, H784Q)CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTA CTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGCTGCAGGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC.SEQ ID NO. 89, amino acid sequence of Mutant ID 18 (V783L, H784Q)MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQ LQ DELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA.

Example 11 BLAST Search for Additional Wild-Type VH-Related DNAPolymerases

A BLAST search using Taq DNA polymerase sequences G755 through P812 (SEQID NO. 90) as a comparison window was performed using available on-linedatabases through the National Center for Biotechnology Information ofthe National Library of Medicine of the National Institutes of Health(http://www.ncbi.nlm.nih.gov). The BLAST search revealed numerouswild-type DNA polymerase from other species sharing extensive sequenceidentity with Taq DNA polymerase, including identity at positions V783and H784 of Taq DNA polymerase (“VH-related DNA polymerases”). Anexemplary listing of these thermostable polymerases is illustrated inTable 24 and similar listing of putatively thermosenstive polymerases isillustrated in Table 25. In all the identified wild-type polymerasegenes except one (Facklamia hominis), the amino acids corresponding toV783 and H784 of Taq DNA polymerase are preserved. In the exceptionalcase, however, namely, Facklamia hominis, an Ile naturally occurs at theresidue position of the Taq DNA polymerase corresponding to V783.However, the Taq DNA polymerase mutant corresponding to Mutant ID 1 thatincludes this particular substitution behaves like the wild-type Taq DNApolymerase. Thus, the DNA polymerase of Facklamia hominis apparentlydeviates from the strong selection of Val at this position is postulatedto maintain wild-type activity if either a Val or Ile residue ispresent. These BLAST results confirm a natural counter-selection againstDNA polymerases having enhanced template discrimination activity andprovide strong evidence that the disclosed engineered Taq DNA polymerasemutants having these properties are novel and non-obvious.

These identified DNA polymerases share extensive sequence homology withTaq DNA polymerase in the region that includes residues V783 and V784 ofTaq DNA polymerase. Like that observed with the engineered Taq DNApolymerase mutants, each of the identified non-Taq DNA polymerasesrepresent a sequence space from which engineered mutant enzymes can begenerated having enhanced template discrimination activity, as comparedto their respective unmodified counterparts. The magnitude of theenhanced template discrimination activity obtained for identical aminoacid substitutions for non-Taq DNA polymerases may not be identical whencompared to the respective unmodified non-Taq DNA polymerases or evenwhen compared to the magnitude of enhanced template discriminationactivity observed for the corresponding Taq DNA polymerase mutant.Nevertheless, a strong prediction of this disclosure is that at leastsome amino acid substitutions in non-Taq DNA polymerases having homologyto residues V783 and/or H784 of Taq DNA polymerase will display enhancedtemplate discrimination activity relative to their respective unmodifiedcounterparts.

TABLE 24Non-Taq thermostable DNA polymerases having homology to Taq sequences in region of V783 and H784SEQ Accession No. Species Alignment (Query: Taq; Sbjct: Species)Identity* ref|WP 018111631.1| Thermus Query 1GTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYP 58 88%SEQ ID NO. 91 igniterraeGTAADLMKLAMV + LFPRL + E + GARMLLQVHDELVLEAPK + RAE VA LAKEVMEGV + PSbjct 752 GTAADLMKLAMVRLFPRLQELGARMLLQVHDELVLEAPKDRAERVAALAKEVMEGVVVP809 ref|WP_022798807.1| Thermus Query 1GTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYP 58 90%SEQ ID NO 92 islandicusGTAADLMKLAMVKLFPRL E GARMLLQVHDEL + LEAPK + RAE VA LAKEVMEGVYP Sbjct 752GTAADLMKLAMVKLFPRLREAGARMLLQVHDELLLEAPKDRAEEVAALAKEVMEGVYP 809ref|YP 005654546.1| Thermus sp. Query 1GTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYP 58 84%SEQ ID NO. 93 CCB_US3_UF1GTAADLMKLAMV + LFP L + GARMLLQVHDEL + LEAPKERAE VARLA + EVMEGV + P Sbjct756 GTAADLMKLAMVRLFPLLPGVGARMLLQVHDELLLEAPKERAEEVARLAREVMEGVVVP 813ref|WP 018461567.1| Thermus Query 1GTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYP 58 83%SEQ ID NO. 94 oshimaiGTAADLMKLAMVKLFPRL + G R + LLQVHDELVLEAPK RAE A + LAKE MEGVYP Sbjct 753GTAADLMKLAMVKLFPRLRPLGVRILLQVHDELVLEAPKARAEEAAQLAKETMEGVYP 810ref|WP 008632471.1| Thermus sp. RL Query 1GTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYP 58 84%SEQ ID NO. 95GTAADLMKLAMVKLFPRL EMGARMLLQVHDEL + LEAP + RAE VA LAKE ME YP Sbjct 754GTAADLMKLAMVKLFPRLREMGARMLLQVHDELLLEAPQARAEEVAALAKEAMEKAYP 811ref|YP 005640602.1| Thermus Query 1GTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYP 58 84%SEQ ID NO: 96 thermophilus SG0.5JP17-16GTAADLMKLAMVKLFPRL EMGARMLLQVHDEL + LEAP + RAE VA LAKE ME YP Sbjct 754GTAADLMKLAMVKLFPRLREMGARMLLQVHDELLLEAPQARAEEVAALAKEAMEKAYP 811ref|WP 019550117.1|\ Thermus Query 1GTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYP 58 83%SEQ ID NO. 97 scotoductus GTAADLMKLAMVKLFPRL + E + GARMLLQVHDELVLEAPKE + AE VA + AK ME V + P Sbjct753 GTAADLMKLAMVKLFPRLQELGARMLLQVHDELVLEAPKEQAEEVAQEAKRTMEEVVVP 810gb|AAB81398.1| Thermus Query 1GTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYP 58 83%SEQ ID NO. 98 caldophilus GTAADLMKLAMVKLFPRL EMGARMLLQVHDEL + LEAP + AE VA LAKE ME YP Sbjct 757GTAADLMKLAMVKLFPRLREMGARMLLQVHDELLLEAPQAGAEEVAALAKEAMEKAYP 814ref|YP 004367987.1| Marini-thermus Query 1GTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVY 57 75%SEQ ID NO. 99 hydro-thermalis DSM 14884GTAADLMKLAMVKL P + + GAR + + LQVHDELVLEAP + ERAEAVAR + + EVMEG + Sbjct758 GTAADLMKLAMVKLAPEIRSLGARLILQVHDELVLEAPQERAEAVARVVREVMEGAW 814gb|AAR11876..1| Thermus Query 1GTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYP 58 72%SEQ ID NO. 100 filiformisGTAADLMK + AMVKLFPRL + + GA + LLQVHDELVLE P + + RAE L KEVME YP Sbjct 755GTAADLMKIAMVKLFPRLKPLGAHLLLQVHDELVLEVPEDRAEEAKALVKEVMENTYP 812ref|WP 0184.65880.1| Meiothermus Query 1GTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVY 57 68%SEQ ID NO. 101 timidusGTAADLMKLAMVKL P+ LE + A + + LQVHDELV + EAP + ERAE VA LA + E M Sbjct 774GTAADLMKLAMVKLGPKLEPLDAHLVLQVHDELVIEAPRERAEEVAELARETMRTAW 830ref|WP 013637959.1| Desulfuro-bacterium Query 1GTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGV 56 61%SEQ ID NO. 102 thermolithot rophumGTAAD+MKLAMVKL + + LE + + GA M + LQVHDE + V + EA + E + E + + + KE ME VSbjct 765 GTAADIMKLAMVKLYKKLEKLGAYMVLQVHDEIVIEALEEKTEEIMKIVKETMENV 820ref|YP 005442159.1| Caldilinea Query 1GTAADLMKLAMVKLFPRLEEMG--ARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVY 57 54%SEQ ID NO. 103 aerophilaGTAAD + MK + AM + + L + RL + G R + L + QVHDELVLEAP E E + L + E M Y Sbjct 875 GTAADIMKIAMIRLYERLQNDGYRTRLLIQVHDELVLEAPPEELESATHLVRETMANAY933 *Sequence identity refers to the percent identity of the querysequence with wild type Taq DNA polymerase.

TABLE 25Non-Taq putatively thermosensitive DNA polymerasess having homology to Taq sequence in theregion of V783 and H784 Acces- SEQ sion Iden- No. SpeciesAlignment (Query: Taq; Sbjct: Species) tity* ref| Eubacterium Query 1GTAADLMKLAMVKLFPRLEEMG--ARMLLQVHDELVLEAPKERAEAVARLAKEVMEG 55 60% WP siraeum GTAAD + + K + AM + K + + RLEE G AR + + LQVHDEL + +  015519EA + + AE VA L KE ME 435.1| Sbjct 751GTAADIIKIAMIKVYNRLEESGLDARLILQVHDELIVEAKEDCAEKVALLLKEEMEN 807 SEQ ID NO: 104 ref| Clostridium Query 1GTAADLMKLAMVKLFPRL--EEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEG 55 58% WP leptum GTAAD + + K + AMV + + RL E M AR + + LQVHDEL + + EAP + +   022236AE AR + E MEG 670.1| Sbjct 320GTAADIIKIAMVRVDRRLKRENMRARLILQVHDELIVEAPEDEAEQAARILTEEMEG 376 SEQ ID NO: 105 ref| Entero- Query 1GTAADLMKLAMVKLFPRLEEMG--ARMLLQVHDELVLEAPKERAEAVARLAKEVME 54 59% WP coccus G + AAD + + K + AM + + L RL + E G A MLLQVHDELV E PK +  002333E + + + L KEVME 048.1| Sbjct 803GSAADILKIAMIELDKRLKETGLQATMLLQVHDELVFEVPKKELESLDKLVKEVME 858 SEQ ID NO: 106 ref| Facklamia Query 1GTAADLMKLAMVKLFPRLEEMG--ARMLLQVHDELVLEAPKERAEAVARLAKEVMEG 55 60% WP hominis GTAAD + + KLAMV + L RLEE G + R + LLQ + HDEL + LE PKE + +  016648L EVME 372.1| Sbjct 803GTAADIIKLAMVRLQARLEEAGLSSRLLLQIHDELILEGPKEEMPQLQKLVVEVMES 859 SEQ ID NO: 107 Ref| Bacillus Query 1GTAADLMKLAMVKLFPRLEEMG--ARMLLQVHDELVLEAPKERAEAVARLAKEVME 54 61% WP anthmcis GTAAD + + K AM + + RLEE G AR + LLQVHDEL + EAPKE E + + L  00041EVME 2792| Sbjct 799GTAADIIKKAMIIMADRLEEEGLQARLLLQVHDELIFEAPKEEVEKLEKLVPEVME 854 SEQ ID NO: 108 ref| Bacillus Query 1GTAADLMKLAMVKLFPRLEEMG--ARMLLQVHDELVLEAPKERAEAVARLAKEVME 54 61% NP 9810cereus GTAAD + + K AM + + RLEE G AR + LLQVHDEL + EAPKE E + + L  11.1|ATCC EVME SEQ ID  10987 Sbjct 799GTAADIIKKAMIIMADRLEEEGLQARLLLQVHDELIFEAPKEEIEKLEKLVPEVME 854 NO: 109*Sequence identity refers to the percent identity of the query sequencewith wild type Taq DNA polymerase.

Example 12 Production of Additional Codon Optimized Taq DNA PolymeraseMutants at Position H784

After determining the properties of the first eighteen mutant versionsof the Taq polymerase (Table 3, Mut IDs 1-18), an additional eighteenmutant versions of Taq DNA polymerase (Table 3, Mut IDs 19-30) were madeby site directed mutagenesis of the cloned OptiTaq codon-optimized WTTaq DNA polymerase. The full set represents all possible amino acidvariations at position 784 in Taq polymerase. Specific mutations wereintroduced into the OptiTaq sequence using the method of PCRsite-directed mutagenesis (Weiner M P, et al., Gene. 151(1-2):119-23(1994)). Each mutagenesis reaction employed 10 pmoles of twocomplementary oligonucleotides (Table 26) containing the desired basechanges, annealed to the double-stranded OptiTaq plasmid (20 ng), 5 UKOD DNA polymerase (Novagen-EMD Chemicals, San Diego, Calif.), 1.5 mMMgSO₄, in 1× KOD PCR buffer. Thermal cycling parameters were 95° C. for3 minutes (95° C. for 20 sec-55° C. for 20 sec-70° C. for 2.5 minutes)for 16 cycles followed by a 70° C. soak for 4 minutes. After PCRsite-directed mutagenesis, the amplified product was treated with 10 Uof Dpn I (NEB, Ipswich, Mass.), at 37° C. for 1 hour, followed byinactivation at 80° C. for 20 minutes. 1/110^(th) of the digestionmaterial was transformed into XL-1 Blue competent bacteria. Bacterialclones were isolated, plasmid DNA prepared, and individual mutationswere confirmed by Sanger DNA sequencing. All mutants remained in thepET-27b(+) expression vector, which is suitable for expressing therecombinant proteins in E. coli. Expression and purification of therecombinant mutants of the Taq polymerase were performed as described inExample 3.

TABLE 26 Oligonucleotides used for site-directed mutagenesis to produce 18 Taq DNA Polymerase mutants at position 784. AminoSequence″ SEQ Sequence″ SEQ Mutant acid Sense mutagenesis IDAntisense mutagenesis ID ID changes oligonucleotide No. oligonucleotideNo. 19 H784G gggcgcacgtatgcttctgca 110 taggggcttctaacaccagctcg 111ggtcGGTgacgagctggtgtt tcACCgacctgcagaagcatacg agaagccccta tgcgccc 20H784A gggcgcacgtatgcttctgca 112 taggggcttctaacaccagctcg 113ggtcGCGgacgagctggtgtt tcCGCgacctgcagaagcatacg agaagccccta tgcgccc 21H784S gggcgcacgtatgcttctgca 114 taggggcttctaacaccagctcg 115ggtcAGCgacgagctggtgtt tcGCTgacctgcagaagcatacg agaagccccta tgcgccc 22H784T gggcgcacgtatgcttctgca 116 taggggcttctaacaccagctcg 117ggtcACGgacgagctggtgtt tcCGTgacctgcagaagcatacg agaagccccta tgcgccc 23H784C gggcgcacgtatgcttctgca 118 taggggcttctaacaccagctcg 119ggtcTGCgacgagctggtgtt tcGCAgacctgcagaagcatacg agaagccccta tgcgccc 24H784V gggcgcacgtatgcttctgca 120 taggggcttctaacaccagctcg 121ggtcGTAgacgagctggtgtt tcTACgacctgcagaagcatacg agaagccccta tgcgccc 25H784L gggcgcacgtatgcttctgca 122 taggggcttctaacaccagctcg 123ggtcTTGgacgagctggtgtt tcCAAgacctgcagaagcatacg agaagccccta tgcgccc 26H784I gggcgcacgtatgcttctgca 124 taggggcttctaacaccagctcg 125ggtcATTgacgagctggtgtt tcAATgacctgcagaagcatacg agaagccccta tgcgccc 27H784M gggcgcacgtatgcttctgca 126 taggggcttctaacaccagctcg 127ggtcATGgacgagctggtgtt tcCATgacctgcagaagcatacg agaagccccta tgcgccc 28H784P gggcgcacgtatgcttctgca 128 taggggcttctaacaccagctcg 129ggtcCCAgacgagctggtgtt tcTGGgacctgcagaagcatacg agaagccccta tgcgccc 29H784F gggcgcacgtatgcttctgca 130 taggggcttctaacaccagctcg 131ggtcTTTgacgagctggtgtt tcAAAgacctgcagaagcatacg agaagccccta tgcgccc 30H784Y gggcgcacgtatgcttctgca 132 taggggcttctaacaccagctcg 133ggtcTATgacgagctggtgtt tcATAgacctgcagaagcatacg agaagccccta tgcgccc 31H784W gggcgcacgtatgcttctgca 134 taggggcttctaacaccagctcg 135ggtcTGGgacgagctggtgtt tcCCAgacctgcagaagcatacg agaagccccta tgcgccc 32H784D gggcgcacgtatgcttctgca 136 taggggcttctaacaccagctcg 137ggtcGATgacgagctggtgtt tcATCgacctgcagaagcatacg agaagccccta tgcgccc 33H784E gggcgcacgtatgcttctg 138 taggggcttctaacaccagctcg 139caggtcGAAgacgagctgg tcTTCgacctgcagaagcatacg tgttagaagccccta tgcgccc 34H784N gggcgcacgtatgcttctgca 140 taggggcttctaacaccagctcg 141ggtcAACgacgagctggtgtt tcGTTgacctgcagaagcatacg agaagccccta tgcgccc 35H784K gggcgcacgtatgcttctgca 142 taggggcttctaacaccagctcg 143ggtcAAAgacgagctggtgtt tcTTTgacctgcagaagcatacg agaagccccta tgcgccc 36H784R gggcgcacgtatgcttctgca 144 taggggcttctaacaccagctcg 145ggtcCGGgacgagctggtgtt tcCCGgacctgcagaagcatacg agaagccccta tgcgccc DNAbases identical to codon optimized OptiTaq are shown in lower case;those specific for the mutations introduced by site-directed mutagenesisare shown in upper case.

Example 13 Characterization of Properties of 18 Mutant Taq DNAPolymerases Altered at Position H784 in PCR

The 18 mutant Taq DNA polymerase enzymes described in Example 12 werecharacterized for polymerase activity and ability to discriminate a3′-RNA residue in the primer oligonucleotide.

The unit activity of the purified wild-type protein was determined bycomparing performance in qPCR of known quantities of OptiTaq and eachmutant compared to a commercial native non-hot-start Taq DNA polymerase,Taq-B DNA Polymerase (Enzymatics, Beverly, Mass.). Quantification cyclevalues (Cq, the amplification cycle number at which positive signal isfirst detected) and amplification curve shapes were analyzed todetermine the nanogram amounts at which both enzymes performed similarlyin the suboptimal range for each. Using these nanogram amounts and knownunit values of Taq-B DNA polymerase, relative activity unit values couldbe extrapolated for all of the mutant DNA polymerase enzymes havingsufficient activity to support PCR.

The following reaction conditions were employed: 1× qPCR buffer (20 mMTris pH 8.4, 50 mM KCl, 3 mM MgCl₂, 0.01% Triton-X100), 800 μM dNTPs(200 μM each), 500 nM For primer (Hs HPRT F517, SEQ ID NO. 43), 500 nMRev primer (Hs HPRT R591, SEQ ID NO. 44), 250 nM probe (Hs HPRT P554,SEQ ID NO. 45), 2×10³ copies of linearized cloned plasmid template(HPRT-targ, SEQ ID NO. 46), in 10 μL final volume. The amount of DNApolymerase added to each reaction was varied as follows: for wild type(OptiTaq), reactions were set using 10, 1, 0.1, 0.01, and 0.001 U/μL(220, 22, 2.2, 0.22, or 0.022 ng of protein per 10 μL reaction). Mutantpolymerases were run in similar concentrations. In addition, thosemutant enzymes showing polymerase activity were more finely titratedtesting 220, 22, 10.6, 4.8, 2.2, 1.1, 0.48, and 0.22 ng of protein per10 μL reaction. Enzyme dilutions were made in enzyme dilution buffer (20mM Tris pH7.5, 100 mM NaCl, 1 mM DTT, 0.1% Triton-X100, 1 mg/mL BSA, 10%glycerol). Reactions were run in 384 well format on a BIO-RAD CFX384™Real-Time System (BIO-RAD, Hercules, Calif.) using cycling parameters95° C. for 30 seconds followed by 60 cycles of [95° C. for 15 secondsfollowed by 60° C. for 1 minutes]. Detection was achieved using afluorescence-quenched probe (5′-nuclease assay format, note that themutations introduced into the present series of Taq mutants do not liein the 5′-nuclease domain). Sequences of the primers, probe, andtemplate (plasmid insert) are shown in Table 27.

TABLE 27 Sequence of oligonucleotides employed in Taq DNA polymerase activity assay. Name Sequence SEQ ID NO. Hs HPRTGACTTTGCTTTCCTTGGTCAG SEQ ID NO. 43 F517 Hs HPRT GGCTTATATCCAACACTTCGTGSEQ ID NO. 44 R591 Hs HPRT FAM-ATGGTCAAG(ZEN)GTCG SEQ ID NO. 45 P554CAAGCTTGCTGGT-IBFQ HPRT- GACTTTGCTTTCCTTGGTCAGGCAG SEQ ID NO. 46 targTATAATCCAAAGATGGTCAAGGTCG CAAGCTTGCTGGTGAAAAGGACCCCACGAAGTGTTGGATATAAGCC Nucleic acid sequences are shown 5′-3′. FAM= 6-carboxyfluorescein, IBFQ = Iowa Black FQ (fluorescence quencher),and ZEN = ZEN internal fluorescence quencher.

These 18 Taq DNA polymerase mutants were characterized as outlinedabove. Results are summarized in Table 28. Ten mutants, including MutantIDs 19, 23, 25, 28, and 31 to 36, did not show detectable DNA polymeraseactivity and were not studied further. Four mutants, Mutant IDs 20, 21,27 and 29 had DNA polymerase activity; however, processivity was reducedfrom 4-6 fold relative to the wild type enzyme. Three mutants, MutantIDs 24, 26, and 30, showed DNA polymerase activity similar to wild typeOptiTaq.

TABLE 28 Activity of novel Taq DNA polymerase mutants. Amino acid ΔCqDelay in Mutant changes from Polymerase Relative priming from IDwild-type Taq Activity activity* an RNA base** 19 H784G No — — 20 H784AYes 0.2  1 21 H784S Yes 0.16 2 22 H784T Yes 1   0 23 H784C No — — 24H784V Yes 0.45 >35  25 H784L No — — 26 H784I Yes 0.5  6 27 H784M Yes0.22 >35  28 H784P No — — 29 H784F Yes 0.22 3 30 H784Y Yes 0.45 5 31H784W No — — 32 H784D No — — 33 H784E No — — 34 H784N No — — 35 H784K No— — 36 H784R No — — *Wild-type OptiTaq was set to “1” and the relativeactivity of each of the mutant polymerases was normalized to thisamplification efficiency, with 1 as the maximum. **ΔCq = [Cq Mutant IDX] − [Cq OptiTaq] when qPCR reactions are run using primers having a3′-RNA residue.

The subset of these mutant Taq DNA polymerases which showed suitablelevels of DNA polymerase activity were studied for their ability todiscriminate between primers have a 3′-DNA versus a 3′-RNA residuerelative to the wild type OptiTaq enzyme. Real-time PCR was performed asbefore, employing in the reactions the amount of each mutant DNApolymerase equal to 0.5 units of wild-type OptiTaq per 10 μL reaction.The following reaction conditions were employed: 1× qPCR buffer (20 mMTris pH 8.4, 50 mM KCl, 3 mM MgCl₂, 0.01% Triton-X100), 800 μM dNTPs(200 μM each), 500 nM For primer (Hs SFRS9 F569 rU, SEQ ID NO. 47), 500nM Rev primer (Hs SFRS9 R712 rA, SEQ ID NO. 48), 250 nM probe (Hs SFRS9P644, SEQ ID NO. 49), 2×10³ copies of linearized cloned plasmid template(SFRS9-targ, SEQ ID NO. 50), in 10 μL final volume. Reactions were runin 384 well format on a BIO-RAD CFX384™ Real-Time System (BIO-RAD,Hercules, Calif.) using cycling parameters 95° C. for 30 secondsfollowed by 60 cycles of [95° C. for 15 seconds followed by 60° C. for 1minutes]. Detection was achieved using a fluorescence-quenched probe(5′-nuclease assay format). Sequences of the primers, probe, andtemplate (plasmid insert) are shown in Table 29.

TABLE 29 Sequence of oligonucleotides employed in the primer 3′-RNA discrimination assay. Name Sequence SEQ ID NO.Hs SFRS9 TGTGCAGAAGGATGGAGu SEQ ID NO. 47 F569 rU Hs SFRS9CTGGTGCTTCTCTCAGGATa SEQ ID NO. 48 R712 rA Hs SFRS9 HEX-TGGAATATG(ZEN)CCSEQ ID NO. 48 P644 CTGCGTAAACTGGA-IBFQ SFRS9- TGTGCAGAAGGATGGAGTGGSEQ ID NO. 50 targ GGATGGTCGAGTATCTCAGA AAAGAAGACATGGAATATGCCCTGCGTAAACTGGATGACA CCAAATTCCGCTCTCATGAG GGTGAAACTTCCTACATCCGAGTTTATCCTGAGAGAAGCA CCAG Nucleic acid sequences are shown 5′-3′ withDNA uppercase and RNA lowercase. HEX = hexachlorofluorescein, IBFQ= Iowa Black FQ (fluorescence quencher), and ZEN = ZEN fluorescencequencher.

The eight Taq DNA polymerase mutants that supported PCR were tested forthe ability to use a 3′-RNA modified primer as outlined above. Resultsare summarized in Table 28. Mutant IDs 20 and 22 did not show anysignificant difference between primers having a 3′-DNA versus a 3′-RNAresidue. Mutant IDs 21, 24, 26, 27, 29, and 30 showed an amplificationdelay using 3′-RNA primers. Thus, additional Taq DNA polymerase mutantswere identified which discriminate against priming from a 3′-RNAresidue. Those mutants which showed some delay with RNA priming andshowed high processivity were further studied for improvements in primer3′-residue mismatch discrimination.

Example 14 Improved Mismatch Discrimination in Allele-Specific PCR usingMutant Taq DNA Polymerases Altered at Position H784

Of the 18 mutant enzymes studied in Example 12 and 13, Mutant IDs 21,24, 26, 27, 29, and 30 showed the ability to discriminate against a3′-RNA residue in the primer and retained high enzymaticactivity/processivity. These six mutants and additionally Mutant IDs 20and 22 were studied for the ability to discriminate against a3′-terminal DNA mismatch compared with wild type OptiTaq DNA polymeraseusing an allele-specific qPCR assay. Amplification reactions wereperformed against a synthetic oligonucleotide template where a singlebase was varied (SNP) which was positioned to lie at the 3′-end of thereverse primer. Synthetic templates were employed having each of the 4possible bases at this position. Reverse primers were employed havingeach of the 4 possible bases at the 3′-end. Relative amplificationefficiencies of all pairwise primer/template combinations were assessedusing qPCR.

Quantitative allele-specific real-time PCR (AS-qPCR) was performed in 10μL reaction volumes in 384 well format with 2×10⁵ copies of a 103 bpsynthetic template (SEQ ID NOs. 51-4). Final reaction conditions usedwere 20 mM Tris-HCL (pH 8.4 at 25° C.), 50 mM KCL, and 3 mM MgCl₂, 0.01%Triton X-100, 800 μM total dNTPs, and 200 nM of the universal forwardprimer (SEQ ID NO. 60), 200 nM of a reverse primer (separate reactionswere set up for each of the allele-specific primers SEQ ID NOs. 55-58 orthe control universal primer SEQ ID NO. 59) and 200 nM of the 5′nuclease detection probe (SEQ ID NO. 61). Each allele-specific primerwas tested on each SNP template. Reactions utilized either 0.5 U (10.8ng/11.1 nM/111 fmol) of the wild type OptiTaq DNA polymerase or 0.5 U ofone of the nine Taq DNA polymerase mutants studied (Mutant ID 3 (H784Q)(10.8 ng/11.1 nM/111 fmol); Mutant ID 20 H784A (54 ng/55.5 nM/555 fmol);Mutant ID22 H784T (10.8 ng/11.1 nM/111 fmol); Mutant ID 24 H784V (24ng/24.7 nM/246.7 fmol); Mutant ID 26 H784I (21.6 ng/22.2 nM/222 fmol);Mutant ID 27 H784M (10.8 ng/11.1 nM/111 fmol); Mutant ID 29 H784F (49.1ng/49.4 nM/494.5 fmol); Mutant ID 30 H784Y) (24 ng/24.7 nM/246.7 fmol).Amplification was performed on a CFX384™ C1000™ Thermo Cycler system(Bio-Rad, Hercules, Calif.) using the following cycling parameters: 95°C. for 30 seconds initial denaturation followed by 60 cycles of 95° C.for 10 seconds, then 60° C. for 30 seconds. Oligonucleotide reagentsused in this example are shown in Table 30.

TABLE 30 Synthetic  oligonucleotides employed in Example 13. NameSequence (5′-3′) SEQ ID NO. A  AGCTCTGCCCAAAGATTACC SEQ ID NO. 51Template CTGACAGCTAAGTGGCAGTG GAAGTTGGCCTCAGAAGTAG TGGCCAGCTGTGTGTCGGGGAACAGTAAAGGCATGAAGCT CAG C  AGCTCTGCCCAAAGATTACC SEQ ID NO. 52 TemplateCTGACAGCTAAGTGGCAGTG GAAGTTGGCCTCAGAAGTAG TGGCCAGCTGTGTGTCGGGGCACAGTAAAGGCATGAAGCT CAG G  AGCTCTGCCCAAAGATTACC SEQ ID NO. 53 TemplateCTGACAGCTAAGTGGCAGTG GAAGTTGGCCTCAGAAGTAG TGGCCAGCTGTGTGTCGGGGGACAGTAAAGGCATGAAGCT CAG T  AGCTCTGCCCAAAGATTACC SEQ ID NO. 54 TemplateCTGACAGCTAAGTGGCAGTG GAAGTTGGCCTCAGAAGTAG TGGCCAGCTGTGTGTCGGGGTACAGTAAAGGCATGAAGCT CAG Syn Rev T CTGAGCTTCATGCCTTTACT SEQ ID NO. 55GTT Syn Rev C CTGAGCTTCATGCCTTTACT SEQ ID NO. 56 GTC Syn Rev ACTGAGCTTCATGCCTTTACT SEQ ID NO. 57 GTA Syn Rev G CTGAGCTTCATGCCTTTACTSEQ ID NO. 58 GTG Syn Rev  CTGAGCTTCATGCCTTTACT SEQ ID NO. 59 GTSyn For  AGCTCTGCCCAAAGATTACC SEQ ID NO. 60 CTG Syn ProbeFAM-TTCTGAGGC(ZEN)CA SEQ ID NO. 61 ACTTCCACTGCCACTTA- IBFQ DNA bases areuppercase; FAM = 6-carboxyfluorescein; IBFQ = Iowa Black ™ FQfluorescence quencher; ZEN = internal ZEN fluorescence quencher;underlined base indicates the SNP site in the synthetic template DNA.

Initially all reactions were run in triplicate. Similar results wereobtained for all replicates when using the wild type OptiTaq. However,results showed greater variation for the mutant polymerases. To obtainstatistically meaningful results, each reaction was therefore performed24 times for the mutant polymerases and 21 times for the wild typeenzyme. ΔCq values were calculated as the Cq value obtained for eachmismatched base pair minus the Cq value obtained for the matched basepair (ΔCq=Cq mismatch−Cq match). The ΔCq values for all 24 replicateswere averaged and standard deviations were calculated. Results are shownin Table 31 and are graphically summarized in FIGS. 3C, 3D, 3E, 3F, and3G. Note that the reverse primer is the allele-specific primer, so the“Syn Rev T” primer (SEQ ID NO. 55) is the perfect match to the TemplateA (SEQ ID NO. 51), etc.

TABLE 31 ΔCq values for AS-qPCR reactions using WT OptiTaq and H784mutant Taq DNA polymerases. Reverse Primer Template SEQ A C G T DNA IDSEQ ID SEQ ID SEQ ID SEQ ID Polymerase Name NO. NO. 51 NO. 52 NO. 53 NO.54 OptiTaq Syn Rev T 55 — 1.4 +/− 0.1  0.6 +/− 0.2 4.8 +/− 0.2 Syn Rev G58  8.5 +/− 0.2 —  5.9 +/− 0.2 3.5 +/− 0.2 Syn Rev C 56  2.8 +/− 0.2 7.7+/− 0.1 — 3.9 +/− 0.1 Syn Rev A 57  5.3 +/− 0.2 −0.8 +/− 0.1   6.1 +/−0.1 — MUT ID 3 Syn Rev T 55 — 5.7 +/− 0.2  5.7 +/− 0.3 10.8 +/− 0.4 H784Q Syn Rev G 58 14.5 +/− 0.6 — 12.5 +/− 0.5 6.8 +/− 0.2 Syn Rev C 56 8.5 +/− 1.5 10.6 +/− 0.2  — 7.7 +/− 0.3 Syn Rev A 57 10.3 +/− 0.5 4.1+/− 0.1 11.0 +/− 0.7 — MUT ID 20 Syn Rev T 55 — 7.6 +/− 0.3  7.7 +/− 0.412.3 +/− 0.8  H784A Syn Rev G 58 19.1 +/− 6.0 — 14.8 +/− 1.4 6.3 +/− 0.5Syn Rev C 56  9.6 +/− 0.5 12.4 +/− 4.7  — 8.2 +/− 0.4 Syn Rev A 57 14.9+/− 4.4 7.6 +/− 0.2 14.2 +/− 1.9 — MUT ID 21 Syn Rev T 55 — 7.9 +/− 0.519.6 +/− 8.5 8.6 +/− 0.8 H784S Syn Rev G 58 25.8 +/− 9.2 — 23.9 +/− 9.66.6 +/− 0.3 Syn Rev C 56 11.4 +/− 4.0 16.4 +/− 9.2  — 9.1 +/− 1.5 SynRev A 57 23.1 +/− 8.2 8.4 +/− 0.4 22.9 +/− 8.6 — MUT ID 22 Syn Rev T 55— 1.5 +/− 0.3  3.7 +/− 0.3 5.6 +/− 0.3 H784T Syn Rev G 58 13.3 +/− 0.6 —10.7 +/− 0.5 3.9 +/− 0.2 Syn Rev C 56  5.2 +/− 0.3 9.3 +/− 0.5 — 3.3 +/−0.3 Syn Rev A 57  9.8 +/− 0.3 2.4 +/− 0.2 11.0 +/− 0.4 — MUT ID 24 SynRev T 55 — −0.3 +/− 0.2   1.8 +/− 0.2 2.6 +/− 0.2 H784V Syn Rev G 5810.2 +/− 0.2 —  8.4 +/− 0.1 2.8 +/− 0.2 Syn Rev C 56  2.8 +/− 0.1 4.6+/− 0.1 — 1.8 +/− 0.1 Syn Rev A 57  5.4 +/− 0.1 0.2 +/− 0.1  9.2 +/− 0.2— MUT ID 26 Syn Rev T 55 — 0.3 +/− 0.2  3.1 +/− 0.1 2.4 +/− 0.2 H784ISyn Rev G 58 11.3 +/− 0.2 —  9.0 +/− 0.2 3.4 +/− 0.2 Syn Rev C 56  4.3+/− 0.2 6.7 +/− 0.1 — 2.5 +/− 0.2 Syn Rev A 57  6.3 +/− 0.1 0.7 +/− 0.110.0 +/− 0.2 — MUT ID 27 Syn Rev T 55 — 4.5 +/− 0.2  6.9 +/− 0.2 9.6 +/−0.5 H784M Syn Rev G 58 16.7 +/− 3.9 — 13.7 +/− 0.7 6.5 +/− 0.3 Syn Rev C56  9.5 +/− 0.3 11.0 +/− 0.4  — 7.4 +/− 0.3 Syn Rev A 57 12.7 +/− 0.15.1 +/− 0.1 14.0 +/− 2.0 — MUT ID 29 Syn Rev T 55 — 5.6 +/− 0.2  3.5 +/−0.1 7.0 +/− 0.2 H784F Syn Rev G 58 13.3 +/− 0.6 — 10.3 +/− 0.3 3.0 +/−0.2 Syn Rev C 56  8.1 +/− 0.2 9.7 +/− 0.3 — 5.7 +/− 0.2 Syn Rev A 5710.9 +/− 0.3 4.6 +/− 0.2 11.3 +/− 0.4 — MUT ID 30 Syn Rev T 55 — 5.3 +/−0.2  4.9 +/− 0.2 8.8 +/− 0.2 H784Y Syn Rev G 58 15.7 +/− 4.6 — 11.8 +/−0.5 5.5 +/− 0.3 Syn Rev C 56  7.3 +/− 0.2 9.9 +/− 0.3 — 6.0 +/− 0.2 SynRev A 57 10.2 +/− 0.2 4.5 +/− 0.2 10.5 +/− 0.3 — Average ΔCq values areshown, where ΔCq = [Cq mismatch − Cq match], +/− standard deviationcalculated from 96 replicates.

The wild type OptiTaq showed an average ΔCq for AS-qPCR in thissynthetic amplicon system of 4.1 with a range of −0.8 to 8.5. Mutant ID3 (H784Q) showed an average ΔCq of 9.9 with a range of 4.6 to 21.2.Mutant ID 20 (H784A) showed an average ΔCq of 11.2 with a range of 6.3to 14.9. Mutant ID 21 (H784S) showed an average ΔCq of 15.3 with a rangeof 6.6 to 25.8. Mutant ID 22 (H784T) showed an average ΔCq of 6.6 with arange of 1.5 to 13.3. Mutant ID 24 (H784V) showed an average ΔCq of 4.1with a range of −0.3 to 10.2. Mutant ID 26 (H7841) showed an average ΔCqof 5.0 with a range of 0.3 to 11.3. Mutant ID 27 (H784M) showed anaverage ΔCq of 9.8 with a range of 4.5 to 16.7. Mutant ID 29 (H784F)showed an average ΔCq of 7.8 with a range of 3.5 to 13.3. Mutant ID 30(H784Y) showed an average ΔCq of 8.3 with a range of 5.3 to 15.7.Therefore, in nearly all pairwise combinations of 4 template bases and 43′-terminal primer bases, the mutant Taq DNA polymerases of the presentinvention showed greater discrimination to mismatch than did the wildtype OptiTaq DNA polymerase. The magnitude of improvement for eachmismatch pair is defined by the ΔΔCq, which is the difference ofdiscrimination between the mutant and wild type enzymes (ΔΔCq=ΔCqmutant−ΔCq wild type). The ΔΔCq values were calculated and are shown inTable 32.

TABLE 32 ΔΔCq values for AS-qPCR reactions for the H784 mutant Taq DNApolymerases compared with wild type OptiTaq Reverse Primer Template SEQA C G T DNA ID SEQ ID SEQ ID SEQ ID SEQ ID Polymerase Name NO. NO. 51NO. 52 NO. 53 NO. 54 MUT ID Syn Rev T 55 — 4.3 5.1 6.0 NO. 3 Syn Rev G58 6.0 — 6.6 3.3 H784Q Syn Rev C 56 5.7 2.9 — 3.8 Syn Rev A 57 5.0 4.94.9 — MUT ID 20 Syn Rev T 55 — 6.2 7.1 7.5 H784A Syn Rev G 58 10.6  —8.9 2.8 Syn Rev C 56 6.8 4.7 — 4.3 Syn Rev A 57 9.6 8.4 8.1 — MUT ID 21Syn Rev T 55 — 6.5 19.0  3.8 H784S Syn Rev G 58 17.3  — 18.0  3.1 SynRev C 56 8.6 8.7 — 5.2 Syn Rev A 57 17.8  9.2 16.8  — MUT ID 22 Syn RevT 55 — 0.1 3.1 0.8 H784T Syn Rev G 58 4.8 — 4.8 0.4 Syn Rev C 56 2.4 1.6— −0.6  Syn Rev A 57 4.5 3.2 4.9 — MUT ID 24 Syn Rev T 55 — −1.7  1.2−2.2  H784V Syn Rev G 58 1.7 — 2.5 −0.7  Syn Rev C 56 0.0 −3.1  — −2.1 Syn Rev A 57 0.1 1.0 3.1 — MUT ID 26 Syn Rev T 55 — −1.1  2.5 −2.4 H784I Syn Rev G 58 2.8 — 3.1 −0.1  Syn Rev C 56 1.5 −1.0  — −1.4  SynRev A 57 1.0 1.5 3.9 — MUT ID 27 Syn Rev T 55 — 3.1 6.3 4.8 H784M SynRev G 58 8.2 — 7.8 3.0 Syn Rev C 56 6.7 3.3 — 3.5 Syn Rev A 57 7.4 5.97.9 — MUT ID 29 Syn Rev T 55 — 4.2 2.9 2.2 H784F Syn Rev G 58 4.8 — 4.4−0.5  Syn Rev C 56 5.3 2.0 — 1.8 Syn Rev A 57 5.6 5.4 5.2 — MUT ID 30Syn Rev T 55 — 3.9 4.3 4.0 H784Y Syn Rev G 58 7.2 — 5.9 2.0 Syn Rev C 564.5 2.2 — 2.1 Syn Rev A 57 4.9 5.3 4.4 — Average ΔΔCq values are shown,where ΔΔCq = [ΔCq mutant − ΔCq wild type], from data in Table 17.

Mutant ID 3 (H784Q) showed an average ΔΔCq of 4.9 compared to wild typeOptiTaq. Mutant ID 20 (H784A) showed an average ΔΔCq of 7.1 compared towild type OptiTaq. Mutant ID 21 (H784S) showed an average ΔΔCq of 11.2compared to wild type OptiTaq. Mutant ID 22 (H784T) showed an averageΔΔCq of 2.5 compared to wild type OptiTaq. Mutant ID 24 (H784V) showedan average ΔΔCq of −0.2 compared to wild type OptiTaq. Mutant ID 26(H7841) showed an average ΔΔCq of 1.0 compared to wild type OptiTaq.Mutant ID 27 (H784M) showed an average ΔΔCq of 5.7 compared to wild typeOptiTaq. Mutant ID 29 (H784F) showed an average ΔΔCq of 3.6 compared towild type OptiTaq. Mutant ID 30 (H784Y) showed an average ΔΔCq of 4.2compared to wild type OptiTaq. Therefore, with the exception of MutantID 24 (H784V), each of the mutant Taq DNA polymerases of the presentinvention showed a significant improvement over wild type OptiTaq inmismatch discrimination. Overall, mutant ID 21 (H784S) showed the bestSNP discrimination within the set of 9 mutant enzymes studied in thisexample using this AS-PCR assay.

Example 15 Improved Mismatch Discrimination in rhPCR using Mutant TaqDNA Polymerases in a Human Genomic DNA SNP Assay

Example 14 demonstrated utility of the novel mutant Taq DNA polymerasesof the present invention in a synthetic amplicon rhPCR SNPdiscrimination assay system. The present Example demonstrates utility ofthe novel mutant Taq DNA polymerases in a human genomic DNA rhPCR SNPdiscrimination assays system, examining a SNP site in the SMAD7 gene(NM_005904, C/T SNP, rs4939827). The assays employed target DNAs GM18562(homozygous C/C) and GM18537 (homozygous T/T) from the Coriell Institutefor Medical Research (Camden, N.J., USA). Two differentblocked-cleavable primer designs were tested, including the generation 1(Gen1) “RDDDDx” primers and the generation 2 (Gen2) “RDxxD” primers(see: US Patent Application 2012/0258455 by Behlke et al., entitled,RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS).

Quantitative real-time rhPCR was performed in 10 μL reaction volumes in384 well format with 20 ng (the equivalent of 6600 copies of target) ofhuman genomic DNA (GM18562 or GM18537). Reactions utilized either 0.5 U(10.8 ng/11.1 nM/111 fmol) of wild type OptiTaq DNA polymerase or 0.5 Uof one of the nine Taq DNA polymerase mutants (MUT ID 3, H784Q; MUT ID20, H784A; MUT ID 21, H784S; MUT ID 22, H784T; MUT ID 24, H784V; MUT ID26, H784I; MUT ID 27 H784M MUT ID 29, H784F; MUT ID 30, H784Y). Finalreaction conditions used were 20 mM Tris-HCL (pH 8.4 at 25° C.), 50 mMKCL, 3 mM MgCl2, 0.01% Triton X-100, 800 μM total dNTPs, 200 nM of aforward primer (SEQ ID NOs. 75-79), 200 nM of the universal reverseprimer (SEQ ID NO. 74), and 200 nM of the SMAD7 probe (SEQ ID NO. 80).Sequence of the 85 bp SMAD7 amplicon is shown as SEQ ID NO. 81. Forwardprimers included RDDDDx configuration Gen1 allele-specific rhPCR primers(SEQ ID NOs. 76 and 77), RDxxD configuration Gen2 allele-specific rhPCRprimers (SEQ ID NOs. 78 and 79) and the control universal forward primer(SEQ ID NO.75) which is not allele specific. Oligonucleotide reagentsemployed in this Example are shown in Table 33. Reactions included 1 μLof P.a. RNase H2 at a concentration of 2.6 mU per 10 μL reaction (5fmoles, 0.5 nM) with the exception of MUT ID 21 (H784S) for which 200 mUper 10 μL (384 fmoles, 38.4 nM) was used for the Gen1 RDDDDx primers andcontrol primer (SEQ ID NOs. 75-77) or 200 mU per 10 μL reaction (384fmoles, 38.4 nM) for the Gen2 RDxxD primers (SEQ ID NOs. 78 and 79).Amplification was performed on a Roche LightCycler® 480 (Roche AppliedScience, Indianapolis, Ind., USA) as follows: 95° C. for 3 minutesfollowed by 95 cycles of 95° C. for 10 seconds and 60° C. for 30seconds. All reactions were performed in triplicate.

TABLE 33 Synthetic  oligonucleotides employed in Example 14. NameSequence (5′-3′) SEQ ID NO. SMAD7 Rev CTCACTCTAAACCCCAGCATT 74 SMAD7 ForCAGCCTCATCCAAAAGAGGAAA 75 SMAD7 For  CAGCCTCATCCAAAAGAGGAAAc 76 rC DDDDxAGGAx SMAD7 For  CAGCCTCATCCAAAAGAGGAAAu 77 rU DDDDx AGGAx SMAD7 For CAGCCTCATCCAAAAGAGGAAAc 78 rC DxxD AxxA SMAD7 For CAGCCTCATCCAAAAGAGGAAAu 79 rU DxxD AxxA SMAD7  FAM-CCCAGAGCTCCCTCAGACT80 probe CCT-IBFQ SMAD7  CAGCCTCATCCAAAAGAGGAAAT 81 targetAGGACCCCAGAGCTCCCTCAGAC TCCTCAGGAAACACAGACAATGC TGGGGTTTAGAGTGAG DNAbases are uppercase and RNA bases are lowercase; FAM= 6-carboxyfluorescein; IBFQ = Iowa Black ™ FQ fluorescence quencher;“x” = C3 Spacer (propanediol). Primer and probe binding sites in theSMAD7 target are underlined.

Results using the Gen1 RDDDDx rhPCR primers are shown in Table 34 usingthe Gen2 RDxxD rhPCR primers are shown in Table 35. Use of the mutantTaq DNA polymerases showed significant improvements in SNPdiscrimination in this human genomic DNA rhPCR assay using the Gen1RDDDDx primers, although amplification efficiency was often reduced, asshown by the increases in the match Cqs. Large improvements indiscrimination were seen using the Gen2 RDxxD primers, althoughamplification efficiency was often lost here as well. The Gen2 RDxxDprimers inherently show greater SNP discrimination and these levels wereincreased so that ΔCq values are in some cases were greater than 40amplification cycles between match and mismatch; this level ofdiscrimination would be “greater than assay” for most users, as qPCRreactions are seldom run for over 45-50 cycles and positive signal wasnot detected in these cases until after 70 cycles (Table 35). Thereforeuse of the new mutant Taq DNA polymerases improves SNP discrimination inrhPCR genotyping assays.

TABLE 34 SNP discrimination of a site in the SMAD7 gene using Gen1RDDDDx primers comparing wild type OptiTaq with four mutant Taq DNApolymerases. Cq Cq SEQ mU RNase Value Value DNA ID H2 per C/C T/TPolymerase For Primer NO. 10 μL rxn DNA DNA ΔCq Wild type SMAD7 For 752.6 24.1 24.9 — OptiTaq SMAD7 For 76 2.6 24.3 36.3 11.9 rC DDDDx SMAD7For 77 2.6 35.1 27.5  7.6 rU DDDDx MUT ID 3 SMAD7 For 75 2.6 26.0 28.1 —H784Q SMAD7 For 76 2.6 29.4 49.1 19.7 rC DDDDx SMAD7 For 77 2.6 48.137.6 10.4 rU DDDDx MUT ID 20 SMAD7 For 75 2.6 31.4 33.9 — H784A SMAD7For 76 2.6 37.4 75.9 38.4 rC DDDDx SMAD7 For 77 2.6 65.4 46.2 19.3 rUDDDDx MUT ID 21 SMAD7 For 75 200 29.7 30.4 — H784S SMAD7 For 76 200 32.046.1 14.1 rC DDDDx SMAD7 For 77 200 47.1 32.8 14.3 rU DDDDx MUT ID 22SMAD7 For 75 2.6 24.2 24.9 — H784T SMAD7 For 76 2.6 25.8 39.0 13.3 rCDDDDx SMAD7 For 77 2.6 37.6 28.8  8.8 rU DDDDx MUT ID 24 SMAD7 For 752.6 24.4 24.1 H784V SMAD7 For 76 2.6 24.7 34.5  9.8 rC DDDDx SMAD7 For77 2.6 36.0 25.6 10.4 rU DDDDx MUT ID 26 SMAD7 For 75 2.6 24.4 24.9H784I SMAD7 For 76 2.6 28.5 40.5 12.0 rC DDDDx SMAD7 For 77 2.6 42.530.6 11.8 rU DDDDx MUT ID 27 SMAD7 For 75 2.6 30.9 30.5 H784M SMAD7 For76 2.6 36.1 58.8 22.7 rC DDDDx SMAD7 For 77 2.6 51.7 37.7 14.0 rU DDDDxMUT ID 29 SMAD7 For 75 2.6 25.8 26.5 H784F SMAD7 For 76 2.6 30.9 50.919.9 rC DDDDx SMAD7 For 77 2.6 46.9 36.2 10.7 rU DDDDx MUT ID 29 SMAD7For 75 2.6 27.3 26.7 H784Y SMAD7 For 76 2.6 31.4 46.6 15.3 rC DDDDxSMAD7 For 77 2.6 50.2 37.1 13.1 rU DDDDx DNA targets included GM18562(homozygous C/C) and GM18537 (homozygous T/T) from the Coriell Institutefor Medical Research. ΔCq = [Cq mismatch − Cq match].

TABLE 35 SNP discrimination of a site in the SMAD7 gene using Gen2 RDxxDprimers comparing wild type OptiTaq with four mutant Taq DNApolymerases. Cq Cq SEQ mU RNase Value Value DNA ID H2 per C/C T/TPolymerase For Primer NO. 10 μL rxn DNA DNA ΔCq Wild type SMAD7 For 75200 24.7 25.1 — OptiTaq SMAD7 For 78 200 24.9 39.6 14.7 rC DxxD SMAD7For 79 200 43.4 26.0 17.4 rU DxxD MUT ID 3 SMAD7 For 75 200 26.5 27.5 —H784Q SMAD7 For 78 200 27.2 56.0 28.8 rC DxxD SMAD7 For 79 200 73.4 37.136.3 rU DxxD MUT ID 20 SMAD7 For 75 200 26.0 26.7 — H784A SMAD7 For 78200 26.1 58.7 32.6 rC DxxD SMAD7 For 79 200 64.1 33.2 31.2 rU DxxD MUTID 21 SMAD7 For 75 200 27.0 27.3 — H784S SMAD7 For 78 200 29.9 69.1 39.3rC DxxD SMAD7 For 79 200 >95 62.6 >32.4  rU DxxD MUT ID 22 SMAD7 For 75200 24.8 25.2 — H784T SMAD7 For 78 200 24.8 45.2 20.4 rC DxxD SMAD7 For79 200 57.5 26.8 30.8 rU DxxD MUT ID 24 SMAD7 For 75 200 25.3 24.8 H784VSMAD7 For 78 200 25.3 39.9 14.6 rC DxxD SMAD7 For 79 200 39.3 24.8 39.3rU DxxD MUT ID 26 SMAD7 For 75 200 24.6 24.8 H784I SMAD7 For 78 200 24.844.0 19.2 rC DxxD SMAD7 For 79 200 46.2 26.9 46.2 rU DxxD MUT ID 27SMAD7 For 75 200 30.0 29.7 H784M SMAD7 For 78 200 31.9 80.1 48.2 rC DxxDSMAD7 For 79 200 83.1 40.6 83.1 rU DxxD MUT ID 29 SMAD7 For 75 200 27.326.1 H784F SMAD7 For 78 200 27.8 51.3 23.6 rC DxxD SMAD7 For 79 200 56.329.1 56.3 rU DxxD MUT ID 30 SMAD7 For 75 200 29.0 28.7 H784Y SMAD7 For78 200 29.2 71.8 42.5 rC DxxD SMAD7 For 79 200 73.5 30.4 73.5 rU DxxDDNA targets included GM18562 (homozygous C/C) and GM18537 (homozygousT/T) from the Coriell Institute for Medical Research. ΔCq = [Cq mismatch− Cq match].

The ΔCq values for the SMAD7 SNP genotyping assays are graphicallysummarized in FIG. 5B and 5C for the Gen1 RDDDDx primers and in FIG. 6Band 6C for the Gen2 RDxxD primers. It is clear that not only do thedifferent mutant Taq DNA polymerases of the present invention haveutility in different amplification assays but that the different mutantsshow varying levels of benefit depending on the nature of the assayused. It is therefore useful to have a collection of mutant polymeraseswhose properties can be matched to different assays/applications so thatmaximal benefit is obtained.

Example 16 Improved Discrimination of Rare Alleles in Genomic DNA usingrhPCR with Mutant Taq DNA Polymerases

Use of the Gen2 RDxxD blocked-cleavable primers in rhPCR can detect thepresence of a SNP at a level of 1:1,000 to 1:10,000 in the background ofwild type genomic DNA using native (wild type) Taq DNA polymerase (see:US Patent Application 2012/0258455 by Behlke et al., entitled, RNASEH-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS). The present exampledemonstrates that the mutant Taq DNA polymerases of the presentinvention improve rare allele discrimination in the rhPCR assay.

Rare allele detection experiments were designed to detect the baseidentity of a SNP site in the SMAD7 gene (NM_005904, C/T SNP, rs4939827)and employed target DNAs GM18562 (homozygous C/C) and GM18537(homozygous T/T) (Coriell Institute for Medical Research, Camden, N.J.,USA). Control reactions were set up using 2 ng (660 copies), 0.2 ng (66copies), or 0.02 ng (6.6 copies) of input matched target DNA. Rareallele detection reactions were set up using 2 ng (660 copies), 0.2 ng(66 copies), or 0.02 ng (6.6 copies) of input matched target DNA of oneallele plus 200 ng (66,000 copies) of the other (mismatched) allele.Background was established in reactions that contained 0 copies ofmatched target DNA plus 200 ng (66,000 copies) of the mismatched targetDNA. Both combinations were tested: GM18562 (C/C) as the rare allele inthe presence of excess GM18537 (T/T) and GM18537 (T/T) as the rareallele in the presence of excess GM18562 (C/C).

Quantitative real-time rhPCR was performed in 10 μL reaction volumes in384 well format. Final reaction conditions used were 10 mM Tris-HCL (pH8.4 at 25° C.), 50 mM KCL, 3.5 mM MgCl₂, 0.01% Triton-X100, 0.8 mMdNTPs, 200 nM of one of the SMAD7 forward primers (SEQ ID NOs. 75, 78,and 79), 200 nM of the SMAD7 reverse primer (SEQ ID NO. 74), and 200 nMof the SMAD7 probe (SEQ ID NO. 80). The 85 bp SMAD7 amplicon defined bythese primers is shown as SEQ ID NO. 81. Note that the forward primerswere either unmodified (control, SEQ ID NO. 75) or were specific for theSMAD7 C-allele (SEQ ID NO. 78) or the SMAD7 T-allele (SEQ ID NO. 79)using blocked-cleavable rhPCR Gen2 RDxxD design. Reactions utilizedeither 0.5 U of the wild type OptiTaq DNA polymerase or 0.5 U of one ofthree example Taq DNA polymerase mutants studied (MUT ID 20(H784A); MUTID 27(H784M); MUT ID 30(H784Y)). Reactions included P. abyssi RNase H2at a concentration of 200 mU per 10 μL reaction (384 fmoles) when usingthe SMAD7 For rC DxxD (SEQ ID NO. 78) primer and control reactions or500-600 mU per 10 μL reaction (960-1152 fmoles) when using the SMAD7 ForrU DxxD (SEQ ID NO. 79) primer. Oligonucleotide reagents used in thisExample are shown in Table 36. Cycling was performed on a RocheLightCycler® 480 (Roche Applied Science, Indianapolis, Ind., USA) asfollows: 95° C. for 3 minutes followed by 65 cycles of 95° C. for 10seconds and 60° C. for 30 seconds. All reactions were performed intriplicate.

TABLE 36 Synthetic  oligonucleotides employed in Example 16. NameSequence (5′-3′) SEQ ID NO. SMAD7 Rev CTCACTCTAAACCCCAGCATT 74 SMAD7 ForCAGCCTCATCCAAAAGAGGAAA 75 SMAD7 For CAGCCTCATCCAAAAGAGGAAAc 78 rC DxxDAxxA SMAD7 For CAGCCTCATCCAAAAGAGGAAAu 79 rU DxxD AxxA SMAD7 FAM-CCCAGAGCTCCCTCAGACT 80 probe CCT-IBFQ SMAD7  CAGCCTCATCCAAAAGAGGAAAT81 target AGGACCCCAGAGCTCCCTCAGAC TCCTCAGGAAACACAGACAATGCTGGGGTTTAGAGTGAG DNA bases are uppercase and RNA bases are lowercase;FAM = 6-carboxyfluorescein; IBFQ = Iowa Black ™ FQ fluorescencequencher; “x” = C3 Spacer (propanediol). Primer and probe binding sitesin the SMAD7 target are underlined.

Results were analyzed and are shown in Table 37. The control columnsshow Cq values for matched primer/target reactions with no mismatchedtarget present and establish a quantification standard curve. MUT ID NO.3, H784Q is included in data analysis for comparison. The rare alleledetection columns show Cq values for detection of 660, 66, 6, or 0(background control) copies of matched primer/target in the presence of66,000 copies of mismatched target. It is generally assumed that atleast a 3 cycle difference (ΔCq=3.0 or greater) between background andpositive signal is needed to call a reaction “positive” for rare alleledetection; a 5 cycle difference (ΔCq=5.0 or greater) is preferred. Inthis system, background is the signal observed when amplification isdone using no input target that is matched to the primer, so signalarises solely from amplification originating off the mismatched target.

Using wild type OptiTaq DNA polymerase, detection of the “C” allele inan excess of “T” background and detection of the “T” allele in an excessof “C” background both met the ΔCq 3.0 and ΔCq 5.0 levels of stringencyto call a 1:1000 rare allele detection event (66 copies of match targetin the presence of 66,000 copies of mismatch target). The 1:10,000reactions (6 copies of match target in the presence of 66,000 copies ofmismatch target) did not meet either of these criteria. Thus rhPCR had a1:1000 rare allele detection limit using wild type OptiTaq in thisgenomic DNA SNP system.

In contrast, rhPCR using each of the four mutants showed a 1:10,000 rareallele detection limit for both the “C” and “T” allele targets with aΔCq stringency cutoff of 3.0. MUT ID 3 (H784Q) showed a 1:10,000 rareallele detection limit for both the “C” and “T” targets in this genomicSNP system for the higher ΔCq stringency cutoff of 5.0. The other threemutant Taq DNA polymerases (MUT ID 20(H784A); MUT ID 27(H784M); MUT ID30(H784Y)) showed a 1:10,000 rare allele detection limit for the “C”allele target with a ΔCq stringency cutoff of 5.0 and a 1:10,000 rareallele detection limit for the “T” allele target with a ΔCq stringencycutoff of 3.0. We therefore conclude that the new mutant Taq DNApolymerases of the present invention provide for improved rare alleledetection reactions using blocked-cleavable primers in rhPCR comparedwith use of the wild type DNA polymerase.

TABLE 37 Rare allele detection using Gen2 RDxxD rhPCR primers comparingwild type OptiTaq with new mutant Taq DNA polymerases 200 ng mismatchedtemplate RNase H2 (66,000 copies of “wild type”) Control DNA SEQ ID per10 660 Match 66 Match 6 Match 0 Match (No mismatched template)Polymerase For Primer NO. μL rxn (1:100) (1:1,000) (1:10,000)(background) 660 Match 66 Match 6 Match 0 Match Wild type SMAD7 For 75200 mU 22.1 21.2 21.2 21.8 27.9 31.3 34.4 >65 OptiTaq SMAD7 For 78 200mU 28.2 31.5 35.1 37.0 28.8 33.3 37.3 >65 rC DxxD SMAD7 For 79 500 mU31.0 34.7 37.7 39.7 31.2 34.6 41.0 >65 rU DxxD MUT ID 3 SMAD7 For 75 200mU 23.5 23.6 24.5 24.1 30.5 33.4 38.0 >65 (H784Q) SMAD7 For 78 200 mU29.8 33.8 37.6 >65 30.5 35.5 39.6 >65 rC DxxD SMAD7 For 79 500 mU 32.937.7 44.0 52.3 30.1 35.9 44.9 >65 rU DxxD MUT ID 20 SMAD7 For 75 200 mU23.5 24.1 24.7 24.9 31.6 36.1 40.2 >65 (H784A) SMAD7 For 78 200 mU 31.336.7 43.2 55.3 33.5 39.5 44.7 >65 rC DxxD SMAD7 For 79 500 mU 35.7 40.043.8 54.7 33.3 38.8 41.5 >65 rU DxxD MUT ID 27 SMAD7 For 75 200 mU 24.224.8 25.4 26.4 31.6 36.2 40.2 >65 (H784M) SMAD7 For 78 200 mU 33.7 38.542.0 54.4 33.6 37.8 41.7 >65 rC DxxD SMAD7 For 79 600 mU 39.7 43.2 45.850.0 31.9 36.5 41.0 >65 rU DxxD MUT ID 30 SMAD7 For 75 200 mU 24.8 24.924.8 26.3 38.6 38.9 46.9 >65 (H784Y) SMAD7 For 78 200 mU 28.8 32.9 37.546.5 29.5 33.4 37.8 >65 rC DxxD SMAD7 For 79 500 mU 39.9 45.8 54.6 57.037.7 44.6 53.1 >65 rU DxxD Cq values are shown. For the rare alleledetection series (selective detection of 6-660 copes one genotype in thepresence of 66,000 copies of the other genotype), those reactions havinga ΔCq of 3.0 or better are highlighted in bold font and those having aΔCq of 5.0 or better are highlighted in bold font with underline. ΔCq =[(Cq 0 copies match) − (Cq 6 copies match)], or ΔCq = [(Cq 0 copiesmatch) − (Cq 66 copies match)], or ΔCq = [(Cq 0 copies match) − (Cq 660copies match)].

Example 17 Sequence of Taq DNA Polymerase Mutants Showing ImprovedDiscrimination for Mismatch or the Presence of an RNA Residue at the3′-End of the Primer

The complete amino acid and nucleotide sequences of the codon optimizedmutant enzymes employed in Examples 11-15 are shown below. Althoughthese sequences are easily derived from information provided in Tables1, 3, 4 and 26 by one with skill in the art, the final assembledsequences are provided below for clarity. Base changes are identified inbold underlined font for the nucleic acid and amino acid substitutions.

SEQ ID NO. 146, nucleotide sequence of  Mutant ID 20 (H784A)CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGG TC GCGGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAG CGGCCGC.SEQ ID NO. 147, amino acid sequence of  Mutant ID 20 (H784A)MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQV A DELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA.SEQ ID NO. 148, nucleotide sequence of  Mutant ID 21 (H784S)CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGG TC AGCGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAG CGGCCGC.SEQ ID NO. 149, amino acid sequence of  Mutant ID 21 (H784S)MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQV S DELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA.SEQ ID NO. 150, nucleotide sequence of  Mutant ID 22 (H784T)CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGG TC ACGGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAG CGGCCGC.SEQ ID NO. 151, amino acid sequence of  Mutant ID 22 (H784T)MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQV T DELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA.SEQ ID NO. 152, nucleotide sequence of  Mutant ID 24 (H784V)CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGG TC GTAGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAG CGGCCGC.SEQ ID NO. 153, amino acid sequence of  Mutant ID 24 (H784V)MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQV V DELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA.SEQ ID NO. 154, nucleotide sequence of  Mutant ID 26 (H784I)CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGG TC ATTGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAG CGGCCGC.SEQ ID NO. 155, amino acid sequence of  Mutant ID 26 (H784I)MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQV V DELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA.SEQ ID NO. 156, nucleotide sequence of  Mutant ID 27 (H784M)CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGG TC ATGGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAG CGGCCGC.SEQ ID NO. 157, amino acid sequence of  Mutant ID 27 (H784M)MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQV M DELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA.SEQ ID NO. 158, nucleotide sequence of  Mutant ID 29 (H784F)CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGG TC TTTGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAG CGGCCGC.SEQ ID NO. 159, amino acid sequence of  Mutant ID 29 (H784F)MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQV F DELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA.SEQ ID NO. 160, nucleotide sequence of  Mutant ID 30 (H784Y)CATATGCGTGGTATGCTGCCGTTGTTCGAGCCTAAAGGCCGCGTACTGTTAGTCGATGGTCATCACTTGGCCTATCGGACGTTCCATGCACTCAAAGGTCTGACGACCAGTCGTGGCGAACCGGTCCAGGCTGTTTATGGTTTCGCTAAGTCTTTGCTCAAAGCACTGAAAGAAGACGGGGACGCGGTAATTGTTGTATTTGATGCCAAAGCACCGAGCTTCCGCCACGAAGCTTATGGTGGCTACAAGGCAGGACGCGCCCCTACCCCAGAAGATTTCCCCCGTCAGCTGGCATTAATTAAGGAGTTAGTAGACCTTCTCGGCTTAGCGCGTCTGGAAGTTCCGGGTTATGAGGCGGACGATGTCCTTGCATCCTTGGCTAAAAAGGCCGAAAAAGAGGGCTACGAAGTCCGCATCTTGACGGCAGACAAAGATCTGTACCAGCTTCTGTCTGACCGTATTCATGTTTTGCACCCTGAAGGCTACTTAATCACTCCGGCCTGGCTCTGGGAAAAGTACGGTCTGCGTCCCGATCAGTGGGCGGATTATCGGGCTTTGACGGGAGATGAGAGCGACAACCTGCCAGGAGTTAAGGGCATTGGTGAAAAAACCGCACGTAAGCTGCTTGAAGAGTGGGGTTCCCTGGAAGCCTTGTTAAAAAATCTGGATCGTCTCAAGCCCGCAATTCGTGAAAAGATCCTGGCTCATATGGACGATCTTAAATTAAGTTGGGACCTGGCCAAGGTGCGCACCGATTTACCGCTTGAAGTGGATTTTGCAAAACGCCGTGAGCCGGACCGGGAACGTTTACGCGCTTTCTTAGAGCGTCTGGAATTCGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGG TC TATGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAG CGGCCGC.SEQ ID NO. 161, amino acid sequence of  Mutant ID 30 (H784Y)MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQV Y DELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKEAA.

Example 18 Production of Codon Optimized Taq DNA Polymerase MutantsModified to Eliminate 5′Exonuclease Activity

Additional Taq DNA Polymerase mutants were made that eliminated the 5′exonuclease activity of several of the mutants from Table 3. Taq DNAPolymerase missing the 5′-exonuclease activity was previously named“KlenTaq” (Barnes, W. M., Gene 112:29-35, 1992). Deletion of theN-terminal 5′ exonuclease domain of Taq polymerase improves the mismatchdiscrimination properties of the enzyme (Barnes, W. M., Gene 112:29-35,1992). The present study characterized whether specificity improvementsseen in the Taq DNA Polymerase mutants of the present invention werecombined with mutations which eliminated 5′-exonuclease activity. Theexamples shown here are meant to be exemplary, and in no way limit therange of the claims. Specific mutations were introduced into the OptiTaqsequence using the method of PCR site-directed mutagenesis (Weiner MP,et al., Gene. 151(1-2):119-23 (1994)). Each mutagenesis reactionemployed 10 pmoles of two oligonucleotides (Table 38) to amplify aroundthe plasmid containing the DNA polymerase, excluding the 5′ exonucleasedomain. These primers were manufactured to contain a 5′ phosphate, whichallowed for re-ligation after amplification. Briefly, these primers wereannealed to the double-stranded plasmid containing previouslycharacterized mutant DNA polymerases (MUT IDs 2, 3, 10, 18, 21, and 30)(20 ng each), 5 U KOD DNA polymerase (Novagen-EMD Chemicals, San Diego,Calif.), 1.5 mM MgSO₄, in 1× KOD PCR buffer. Thermal cycling parameterswere 95° C. for 3 minutes (95° C. for 20 sec-55° C. for 20 sec-70° C.for 2 minutes) for 25 cycles followed by a 70° C. soak for 4 minutes.After PCR site-directed mutagenesis, the amplified product was treatedwith 10 U of Dpn I (NEB, Ipswich, Mass.), at 37° C. for 1 hour, followedby inactivation at 80° C. for 20 minutes. ⅙^(th) of the digestionmaterial was ligated together with T4 DNA ligase (NEB, Ipswich, Mass.)at 16° C. for 20 minutes, followed by inactivation at 65° C. for 10minutes. 1/15^(th) of the ligated material was transformed into XL-1Blue competent bacteria. Bacterial clones were isolated, plasmid DNAprepared, and deletion of the 5′ exonuclease domains were confirmed bySanger DNA sequencing. All mutants remained in the pET-27b(+) expressionvector, which is suitable for expressing the recombinant proteins in E.coli. Expression and purification of the recombinant mutants of the Taqpolymerase were performed as described in Example 3.

TABLE 38 Oligonucleotides used for site-directed mutagenesis to produce 18 Taq DNA Polymerase mutants. Sequence″ SEQSequence″ SEQ Mutant Mutant Sense mutagenesis ID Antisense mutagenesisID ID name oligonucleotide No. oligonucleotide No. 37 OptiTaq Phos- 162Phos- 163 KlenTaq ggttcactgcttcatgaattc catatgtattctccttcttaa ggtcagttaaacaaa 38 A661E, Phos- 162 Phos- 163 I665W, ggttcactgcttcatgaattccatatgtattctccttcttaa F667L ggtc agttaaacaaa KlenTaq 39 V783F Phos- 162Phos- 163 KlenTaq ggttcactgcttcatgaattc catatgtattctccttcttaa ggtcagttaaacaaa 40 H784Q Phos- 162 Phos- 163 KlenTaq ggttcactgcttcatgaattccatatgtattctccttcttaa ggtc agttaaacaaa 41 V783L Phos- 162 Phos- 163H784Q ggttcactgcttcatgaattc catatgtattctccttcttaa KlenTaq ggtcagttaaacaaa 42 H784S Phos- 162 Phos- 163 KlenTaq ggttcactgcttcatgaattccatatgtattctccttcttaa ggtc agttaaacaaa 43 H784Y Phos- 162 Phos- 163KlenTaq ggttcactgcttcatgaattc catatgtattctccttcttaa ggtc agttaaacaaa DNAbases identical to codon optimized OptiTaq are shown in lower case;those specific for the mutations introduced by site-directed mutagenesisare shown in upper case.

Example 19 Characterization of Properties of 7 5′-Exonuclease-DeficientMutant Taq DNA Polymerases in PCR

The 7 mutant Taq DNA polymerase enzymes described in Example 18 werecharacterized for polymerase activity.

The unit activity of the purified wild-type protein was determined bycomparing performance in qPCR of known quantities of OptiTaq and eachmutant compared to a commercial non-hot-start Taq DNA polymerase, Taq-BDNA Polymerase (Enzymatics, Beverly, Mass.). Quantification cycle values(Cq, the amplification cycle number at which positive signal is firstdetected) and amplification curve shapes were analyzed to determine thenanogram amounts at which both enzymes performed similarly in thesuboptimal range for each. Using these nanogram amounts and known unitvalues of Taq-B DNA polymerase, relative activity unit values could beextrapolated for all of the mutant DNA polymerase enzymes havingsufficient activity to support PCR. Testing was also done to determinethe MgCl₂ concentrations at which the polymerases would show optimalactivity.

The following reaction conditions were employed: 1× qPCR buffer (20 mMTris pH 8.4, 50 mM KCl, 0.01% Triton-X100), 800 μM dNTPs (200 μM each),500 nM For primer (Hs HPRT F517, SEQ ID NO. 43), 500 nM Rev primer (HsHPRT R591, SEQ ID NO. 44), 250 nM RNase H2 cleavable probe (Hs HPRT RN2Probe, SEQ ID NO. 164), 20 mU Pyrococcus abyssi RNase H2, 2×10³ copiesof linearized cloned plasmid template (HPRT-targ, SEQ ID NO. 46), in 10μL final volume. MgC₂ was tested at 3, 4, or 5 mM in each case. Theamount of DNA polymerase added to each reaction was varied as follows:for wild type (OptiTaq), reactions were set using 10, 1, 0.1, 0.01, and0.001 U/μL (220, 22, 2.2, 0.22, or 0.022 ng of protein per 10 μLreaction). Mutant polymerases were run in similar concentrations. Inaddition, those mutant enzymes showing polymerase activity were morefinely titrated testing 220, 22, 10.6, 4.8, 2.2, 1.1, 0.48, and 0.22 ngof protein per 10 μL reaction. Polymerase dilutions were made in enzymedilution buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1%Triton-X100, 1 mg/mL BSA, 10% glycerol). Reactions were run in 384 wellformat on a BIO-RAD CFX384™ Real-Time System (BIO-RAD, Hercules, Calif.)using cycling parameters 95° C. for 30 seconds followed by 60 cycles of[95° C. for 15 seconds followed by 60° C. for 1 minutes]. Detection wasachieved using a fluorescence-quenched probe (cleaved by the action ofthe P.a. RNase H2 enzyme). Sequences of the primers, probe, and template(plasmid insert) are shown in Table 39.

TABLE 39 Sequence of oligonucleotides employed in Taq DNA polymerase activity assay. Name Sequence SEQ ID NO. Hs HPRTGACTTTGCTTTCCTTGGTCAG SEQ ID NO. 43 F517 Hs HPRT GGCTTATATCCAACACTTCGTGSEQ ID NO. 44 R591 Hs HPRT FAM-ATGGTCAAGGTCGCAAGc SEQ ID NO.  RN2TTGCTGGT-IBFQ 164 Probe HPRT- GACTTTGCTTTCCTTGGTCAGGCAG SEQ ID NO. 46targ TATAATCCAAAGATGGTCAAGGTCG CAAGCTTGCTGGTGAAAAGGACCCCACGAAGTGTTGGATATAAGCC DNA bases are uppercase and RNA bases arelowercase; Nucleic acid sequences are shown 5′-3′. FAM= 6-carboxyfluorescein, IBFQ = Iowa Black FQ (fluorescence quencher),and ZEN = ZEN internal fluorescence quencher.These 7 Taq DNA polymerase 5′-exonuclease-deficient mutants werecharacterized as outlined above. Results are summarized in Table 40. Allseven mutants had DNA polymerase activity; however, processivity inMutant IDs 38, 39, 40, 41, 42, and 43 was reduced from 10-50 foldrelative to the wild type enzyme. One mutant, Mutant ID 37 (OptiTaqKlenTaq), showed DNA polymerase activity nearly identical to wild typeOptiTaq. Therefore the combination of complete deletion of the5′-exonuclease domain of Taq DNA Polymerase coupled with point mutationsthat improve polymerase specificity all significantly compromised enzymeactivity and processivity.

TABLE 40 Novel Taq DNA polymerase mutants selected for initial study.Optimal Amino acid MgCl₂ Mutant changes from Polymerase Relativeconcentration ID wild-type Taq Activity activity* (mM) 37 OptiTaqKlenTaq Yes 1 3 38 A661E, I665W, Yes 0.1 5 F667L KlenTaq 39 V783FKlenTaq Yes 0.05 4 40 H784Q KlenTaq Yes 0.03 4 41 V783L H784Q Yes 0.02 5KlenTaq 42 H784S KlenTaq Yes 0.02 5 43 H784Y KlenTaq Yes 0.05 5*Wild-type OptiTaq was set to “1” and the relative activity of each ofthe mutant polymerases was normalized to this amplification efficiency,with 1 as the maximum.

Example 20 Improved Mismatch Discrimination in Allele-Specific PCR usingMutant Taq DNA Polymerases also having Deletion of the 5′-ExonucleaseDomain

Of the 7 mutant enzymes studied in Example 18 and 19, Mutant IDs 37, 38,39, 40, 41, 42, and 43 retained sufficient enzymaticactivity/processivity to characterize. These seven mutants were studiedfor the ability to discriminate against a 3′-terminal DNA mismatchcompared with wild type OptiTaq DNA polymerase using an allele-specificqPCR assay. Amplification reactions were performed against a syntheticoligonucleotide template where a single base was varied (SNP) which waspositioned to lie at the 3′-end of the reverse primer. Synthetictemplates were employed having each of the 4 possible bases at thisposition. Reverse primers were employed having each of the 4 possiblebases at the 3′-end. Relative amplification efficiency was assessedusing qPCR.

Quantitative allele-specific real-time PCR (AS-qPCR) was performed in 10μL reaction volumes in 384 well format with 2×10⁵ copies of a 103 bpsynthetic template (SEQ ID NOs. 51-4). Final reaction conditions usedwere 20 mM Tris-HCL (pH 8.4 at 25° C.), 50 mM KCL, the amount of MgCl₂which was determined to be optimal for each polymerase in Example 19,0.01% Triton X-100, 800 μM total dNTPs, and 200 nM of the universalforward primer (SEQ ID NO. 60), 200 nM of a reverse primer (separatereactions were set up for each of the allele-specific primers SEQ IDNOs. 55-58 or the control universal primer SEQ ID NO. 59) and 200 nM ofthe RNase H2 cleavable probe (SEQ ID NO. 165). 20 mU Pyrococcus abyssiRNase H2 was also include in each reaction. Each allele-specific primerwas tested on each SNP template. Reactions utilized either 0.5 U (10.8ng/11.1 nM/111 fmol) of the OptiTaq KlenTaq DNA polymerase (Mutant ID37) or 0.5 U of one of the six Taq DNA polymerase mutants studied(Mutant ID 38 (108 ng/111 nM/1110 fmol); Mutant ID 39 (216 ng/222nM/2220 fmol); Mutant ID 40 (360 ng/370 nM/3700 fmol); Mutant ID 41(1060 ng/555 nM/5550 fmol); Mutant ID 42 (1060 ng/555 nM/5550 fmol);Mutant ID 43 (216 ng/222 nM/2220 fmol)). Amplification was performed ona CFX384™ C1000™ Thermo Cycler system (Bio-Rad, Hercules, Calif.) usingthe following cycling parameters: 95° C. for 30 seconds initialdenaturation followed by 60 cycles of 95° C. for 10 seconds, then 60° C.for 30 seconds. Oligonucleotide reagents used in this example are shownin Table 41.

TABLE 41 Synthetic  oligonucleotides employed in Example 20. NameSequence (5′-3′) SEQ ID NO. A  AGCTCTGCCCAAAGATTACC SEQ ID NO. 51Template CTGACAGCTAAGTGGCAGTG GAAGTTGGCCTCAGAAGTAG TGGCCAGCTGTGTGTCGGGGAACAGTAAAGGCATGAAGCT CAG C  AGCTCTGCCCAAAGATTACC SEQ ID NO. 52 TemplateCTGACAGCTAAGTGGCAGTG GAAGTTGGCCTCAGAAGTAG TGGCCAGCTGTGTGTCGGGGCACAGTAAAGGCATGAAGCT CAG G  AGCTCTGCCCAAAGATTACC SEQ ID NO. 53 TemplateCTGACAGCTAAGTGGCAGTG GAAGTTGGCCTCAGAAGTAG TGGCCAGCTGTGTGTCGGGGGACAGTAAAGGCATGAAGCT CAG T  AGCTCTGCCCAAAGATTACC SEQ ID NO. 54 TemplateCTGACAGCTAAGTGGCAGTG GAAGTTGGCCTCAGAAGTAG TGGCCAGCTGTGTGTCGGGGTACAGTAAAGGCATGAAGCT CAG Syn Rev T CTGAGCTTCATGCCTTTACT SEQ ID NO. 55GTT Syn Rev C CTGAGCTTCATGCCTTTACT SEQ ID NO. 56 GTC Syn Rev ACTGAGCTTCATGCCTTTACT SEQ ID NO. 57 GTA Syn Rev G CTGAGCTTCATGCCTTTACTSEQ ID NO. 58 GTG Syn Rev CTGAGCTTCATGCCTTTACT SEQ ID NO. 59 GT Syn ForAGCTCTGCCCAAAGATTACC SEQ ID NO. 60 CTG RN2 Probe FAM-TTCTGAGGCCAACuCCSEQ ID NO.  ACTGCCACTTA-IBFQ 165 DNA bases are uppercase and RNA basesare lowercase; FAM = 6-carboxyfluorescein; IBFQ = Iowa Black ™ FQfluorescence quencher; ZEN = internal ZEN fluorescence quencher;underlined base indicates the SNP site in the synthetic template DNA.

Initially all reactions were run in triplicate. Similar results wereobtained for all replicates when using the wild type OptiTaq. However,results showed greater variation for the mutant polymerases. To obtainstatistically meaningful results, each reaction was therefore performed24 times for the mutant polymerases and 21 times for the wild typeenzyme. ΔCq values were calculated as the Cq value obtained for eachmismatched base pair minus the Cq value obtained for the matched basepair (ΔCq=Cq mismatch−Cq match). The ΔCq values for all 24 replicateswere averaged and standard deviations were calculated. Results are shownin Table 42 and are graphically summarized in FIGS. 7A, 7B, and 7C. Notethat the reverse primer is the allele-specific primer, so the “Syn RevT” primer (SEQ ID NO. 55) is the perfect match to the Template A (SEQ IDNO. 51), etc.

TABLE 42 ΔCq values for AS-qPCR reactions using KlenTaq mutant Taq DNApolymerases. Reverse Primer Template SEQ A C G T DNA ID SEQ ID SEQ IDSEQ ID SEQ ID Polymerase Name NO. NO. 51 NO. 52 NO. 53 NO. 54 Mutant ID37 Syn Rev T 55 —  9.2 +/− 0.3  6.9 +/− 0.4 10.0 +/− 0.4 OptiTaq Syn RevG 58 14.7 +/− 1.2 — 11.3 +/− 0.5  3.9 +/− 0.3 KlenTaq Syn Rev C 56  9.4+/− 0.2 10.4 +/− 0.2 —  7.5 +/− 0.2 Syn Rev A 57 13.3 +/− 0.4  8.7 +/−0.2 12.4 +/− 0.4 — Mutant ID 38 Syn Rev T 55 — 11.8 +/− 0.5  9.9 +/− 0.611.7 +/− 0.7 A661E, Syn Rev G 58 17.4 +/− 5.8 — 13.1 +/− 1.5  7.9 +/−2.9 I665W, Syn Rev C 56 10.8 +/− 0.4 11.0 +/− 0.5 — 10.5 +/− 0.2 F667LSyn Rev A 57 13.8 +/− 0.6 11.5 +/− 0.4 13.6 +/− 0.8 — KlenTaq Mutant ID39 Syn Rev T 55 — 10.9 +/− 0.3  8.7 +/− 0.3 11.3 +/− 0.4 V783F Syn Rev G58 17.1 +/− 6.7 — 12.9 +/− 0.7  6.9 +/− 0.3 KlenTaq Syn Rev C 56 10.5+/− 0.2 10.5 +/− 0.6 — 10.0 +/− 0.2 Syn Rev A 57 13.4 +/− 0.6 10.6 +/−0.2 13.0 +/− 0.4 — Mutant ID 40 Syn Rev T 55 — 11.5 +/− 0.4 10.1 +/− 0.312.0 +/− 0.9 H784Q Syn Rev G 58 18.2 +/− 6.4 — 13.2 +/− 0.5  7.9 +/− 0.3KlenTaq Syn Rev C 56 10.7 +/− 0.4 10.5 +/− 0.6 — 10.2 +/− 0.3 Syn Rev A57 13.8 +/− 0.7 11.3 +/− 0.3 13.5 +/− 0.6 — Mutant ID 41 Syn Rev T 55 — 8.9 +/− 0.3  7.8 +/− 0.3 10.5 +/− 0.4 V783L Syn Rev G 58 15.8 +/− 4.3 —12.4 +/− 0.5  5.8 +/− 0.3 H784Q Syn Rev C 56 10.1 +/− 0.5 10.2 +/− 0.2 — 8.5 +/− 0.4 KlenTaq Syn Rev A 57 13.3 +/− 0.8  9.5 +/− 0.3 12.6 +/− 0.4— Mutant ID 42 Syn Rev T 55 — 12.1 +/− 0.7 10.2 +/− 0.4 11.4 +/− 0.6H784S Syn Rev G 58 15.8 +/− 1.0 — 13.3 +/− 0.6  8.3 +/− 0.5 KlenTaq SynRev C 56 10.3 +/− 0.3 10.6 +/− 0.5 — 10.1 +/− 0.4 Syn Rev A 57 14.1 +/−0.4 12.0 +/− 0.4 14.1 +/− 0.3 — Mutant ID 43 Syn Rev T 55 — 11.3 +/− 0.4 8.5 +/− 0.4 11.0 +/− 0.4 H784Y Syn Rev G 58 15.5 +/− 1.2 — 12.4 +/− 0.7 6.5 +/− 0.3 KlenTaq Syn Rev C 56  9.9 +/− 0.3 10.3 +/− 0.5 —  9.3 +/−0.4 Syn Rev A 57 13.7 +/− 1.2 11.6 +/− 1.2 13.9 +/− 1.5 — Average ΔCqvalues are shown, where ΔCq = [Cq mismatch − Cq match], +/− standarddeviation calculated from 24 replicates.

The OptiTaq KlenTaq Mutant ID 37 showed an average ΔCq for AS-qPCR inthis synthetic amplicon system of 9.8 with a range of 3.9 to 14.7.Mutant ID 38 (A661E, 1665W, F667L KlenTaq) showed an average ΔCq of 11.9with a range of 7.9 to 17.4. Mutant ID 39 (V783F KlenTaq) showed anaverage ΔCq of 11.3 with a range of 6.9 to 17.1. Mutant ID 40 (H784QKlenTaq) showed an average ΔCq of 11.9 with a range of 7.9 to 18.2.Mutant ID 41 (V783L H784Q KlenTaq) showed an average ΔCq of 10.5 with arange of 5.8 to 15.8. Mutant ID 42 (H784S KlenTaq) showed an average ΔCqof 11.9 with a range of 8.3 to 15.8. Mutant ID 43 (H784Y KlenTaq) showedan average ΔCq of 11.2 with a range of 6.5 to 15.5. Therefore, in allpairwise combinations of 4 template bases and 4 3′-terminal primer basesthe mutant Taq DNA polymerases of the present invention showed greaterdiscrimination to mismatch than did the OptiTaq or OptiTaq KlenTaq DNApolymerases. The magnitude of improvement for each mismatch pair isdefined by the ΔΔCq, which is the difference of discrimination betweenthe mutant and wild type KlenTaq enzymes (ΔΔCq=ΔCq mutant KlenTaq−ΔCqOptiTaq KlenTaq). The ΔΔCq values were calculated and are shown in Table43.

TABLE 43 ΔΔCq values for AS-qPCR reactions for the mutant KlenTaq DNApolymerases compared with OptiTaq KlenTaq. Reverse Primer Template SEQ AC G T DNA ID SEQ ID SEQ ID SEQ ID SEQ ID Polymerase Name NO. NO. 51 NO.52 NO. 53 NO. 54 Mutant Syn Rev T 55 — 2.6 3   1.7 ID 38 Syn Rev G 582.7 — 1.8 4 A661E, Syn Rev C 56 1.4 0.6 — 3 I665W, Syn Rev A 57 0.7 2.81.2 — F667L KlenTaq Mutant Syn Rev T 55 — 1.7 1.8 1.3 ID 39 Syn Rev G 582.4 — 1.6 3 V783F Syn Rev C 56 1.1 0.1 — 2.5 KlenTaq Syn Rev A 57 0.31.9 0.6 — Mutant Syn Rev T 55 — 2.3 3.2 2 ID 40 Syn Rev G 58 3.5 — 1.9 4H784Q Syn Rev C 56 1.3 0.4 — 2.7 KlenTaq Syn Rev A 57 0.7 2.6 1.1 MutantSyn Rev T 55 — −0.3  0.9 0.5 ID 41 Syn Rev G 58 1.1 — 1.1 1.9 V783L SynRev C 56 0.7 −0.2  — 1 H784Q Syn Rev A 57 0.2 0.8 0.2 — KlenTaq MutantSyn Rev T 55 — 2.9 3.3 1.4 ID 42 Syn Rev G 58 1.1 — 2   4.4 H784S SynRev C 56 0.9 0.2 — 2.6 KlenTaq Syn Rev A 57 1   3.3 1.7 — Mutant Syn RevT 55 — 2.1 1.6 1 ID 43 Syn Rev G 58 0.8 — 1.1 2.6 H784Y Syn Rev C 56 0.5−0.1  — 1.8 KlenTaq Syn Rev A 57 0.6 2.9 1.5 — Average ΔΔCq values areshown, where ΔΔCq = [ΔCq mutant KlenTaq − ΔCq OptiTaq KlenTaq], fromdata in Table 42.

Mutant ID 38 (A661E, I665W, F667L KlenTaq) showed an average ΔΔCq of 1.7compared to OptiTaq KlenTaq. Mutant ID 39 (V783F KlenTaq) showed anaverage ΔΔCq of 2.0 compared to OptiTaq KlenTaq. Mutant ID 40 (H784QKlenTaq) showed an average ΔΔCq of 2.1 compared to OptiTaq KlenTaq.Mutant ID 41 (V783L H784Q KlenTaq) showed an average ΔΔCq of 0.7compared to OptiTaq KlenTaq. Mutant ID 42 (H784S KlenTaq) showed anaverage ΔΔCq of 2.1 compared to OptiTaq KlenTaq. Mutant ID 43 (H784YKlenTaq) showed an average ΔΔCq of 2.0 compared to OptiTaq KlenTaq.Therefore, each of the mutant Taq DNA polymerases of the presentinvention showed a significant improvement in mismatch discriminationover OptiTaq KlenTaq which had complete deletion of the 5′-exonucleasedomain but contained no other secondary mutations. Overall, mutant IDs40 and 42 (H784Q KlenTaq and H784S KlenTaq) showed the best SNPdiscrimination within the set of mutant enzymes studied in this exampleusing an AS-PCR assay.

Example 21 Improved Mismatch Discrimination in rhPCR using MutantKlenTaq DNA Polymerases in a Human Genomic DNA SNP Assay

Example 20 demonstrated utility of the novel mutant Taq DNA polymerasesof the present invention in a synthetic amplicon rhPCR SNPdiscrimination assay system. The present Example demonstrates utility ofthe novel mutant Taq DNA polymerases in a human genomic DNA rhPCR SNPdiscrimination assays system, examining a SNP site in the SMAD7 gene(NM_005904, C/T SNP, rs4939827). The assays employed target DNAs GM18562(homozygous C/C) and GM18537 (homozygous T/T) from the Coriell Institutefor Medical Research (Camden, N.J., USA). One blocked-cleavable primerdesign was tested, the generation 1 (Gen1) “RDDDDx” primers (see: USPatent Application 2012/0258455 by Behlke et al., entitled, RNASEH-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS).

Quantitative real-time rhPCR was performed in 10 μL reaction volumes in384 well format with 20 ng (the equivalent of 6600 copies of target) ofhuman genomic DNA (GM18562 or GM18537). Reactions utilized either 0.5 U(10.8 ng/11.1 nM/111 fmol) of OptiTaq KlenTaq DNA polymerase or 0.5 U ofone of the three Taq DNA polymerase mutants (Mutant ID 40 (360 ng/370nM/3700 fmol); Mutant ID 41 (1060 ng/555 nM/5550 fmol); Mutant ID 43(216 ng/222 nM/2220 fmol)). Final reaction conditions used were 20 mMTris-HCL (pH 8.4 at 25° C.), 50 mM KCL, 3 mM MgCl₂, 0.01% Triton X-100,800 μM total dNTPs, 200 nM of a forward primer (SEQ ID NOs. 75-79), 200nM of the universal reverse primer (SEQ ID NO. 74), and 200 nM of theRNase H2 cleavable SMAD7 probe (SEQ ID NO. 166). Sequence of the 85 bpSMAD7 amplicon is shown as SEQ ID NO. 81. Forward primers includedRDDDDx configuration Gen1 allele-specific rhPCR primers (SEQ ID NOs. 76and 77), and the control universal forward primer (SEQ ID NO.75) whichis not allele specific. Oligonucleotide reagents employed in thisExample are shown in Table 44. Reactions included 1 μL of P.a. RNase H2at a concentration of 2.6 mU per 10 μL reaction (5 fmoles, 0.5 nM).Amplification was performed on a Roche LightCycler® 480 (Roche AppliedScience, Indianapolis, Ind., USA) as follows: 95° C. for 3 minutesfollowed by 95 cycles of 95° C. for 10 seconds and 60° C. for 30seconds. All reactions were performed in triplicate.

TABLE 44 Synthetic  oligonucleotides employed in Example 21. NameSequence (5′-3′) SEQ ID NO. SMAD7 Rev CTCACTCTAAACCCCAGCATT 74 SMAD7 ForCAGCCTCATCCAAAAGAGGAAA 75 SMAD7 For  CAGCCTCATCCAAAAGAGGAAA 76 rC DDDDxcAGGAx SMAD7 For  CAGCCTCATCCAAAAGAGGAAA 77 rU DDDDx uAGGAx SMAD7 RN2FAM-CCCAGAGCTCcCTCAGAC 166  probe TCCT-IBFQ SMAD7 CAGCCTCATCCAAAAGAGGAAA 81 target TAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAA TGCTGGGGTTTAGAGTGAG DNA bases are uppercase andRNA bases are lowercase; FAM = 6-carboxyfluorescein; IBFQ = Iowa Black™ FQ fluorescence quencher; “x” = C3 Spacer (propanediol). Primer andprobe binding sites in the SMAD7 target are underlined.

Results using the Gen1 RDDDDx rhPCR primers are shown in Table 45. Useof the mutant Taq DNA polymerases showed significant improvements in SNPdiscrimination in this human genomic DNA rhPCR assay using the Gen1RDDDDx primers, although amplification efficiency was often reduced, asshown by the increases in the match Cqs. Therefore use of the new mutantKlenTaq DNA polymerases improves SNP discrimination in rhPCR genotypingassays.

TABLE 45 SNP discrimination of a site in the SMAD7 gene using Gen1RDDDDx primers comparing wild type OptiTaq with four mutant Taq DNApolymerases. Cq Cq SEQ mU RNase Value Value DNA ID H2 per C/C T/TPolymerase For Primer NO. 10 μL rxn DNA DNA ΔCq MUT ID 37 SMAD7 For 752.6 24.4 23.5 OptiTaq SMAD7 For 76 2.6 31.1 38.4 7.3 KlenTaq rC DDDDxSMAD7 For 77 2.6 46.1 33.0 13.1 rU DDDDx MUT ID 40 SMAD7 For 75 2.6 24.624.5 H784Q SMAD7 For 76 2.6 28.1 38.8 10.7 KlenTaq rC DDDDx SMAD7 For 772.6 42.1 28.8 13.3 rU DDDDx MUT ID 41 SMAD7 For 75 2.6 24.5 24.3 V783LSMAD7 For 76 2.6 26.2 37.6 11.4 H784Q rC DDDDx KlenTaq SMAD7 For 77 2.641.2 27.8 13.4 rU DDDDx MUT ID 43 SMAD7 For 75 2.6 24.7 24.8 H784Y SMAD7For 76 2.6 33.8 45.1 11.2 KlenTaq rC DDDDx SMAD7 For 77 2.6 50.4 35.215.2 rU DDDDx DNA targets included GM18562 (homozygous C/C) and GM18537(homozygous T/T) from the Coriell Institute for Medical Research. ΔCq =[Cq mismatch − Cq match].

Example 22 Sequence of Taq DNA Polymerase Mutants Showing ImprovedDiscrimination for Mismatch or the Presence of an RNA Residue at the3′-End of the Primer

The complete amino acid and nucleotide sequences of the codon optimizedmutant enzymes employed in Examples 18-21 are shown below. Althoughthese sequences are easily derived from information provided in Tables1, 3, 4 26 and 38 by one with skill in the art, the final assembledsequences are provided below for clarity. Base changes are identified inbold underlined font for the nucleic acid and amino acid substitutions.

SEQ ID NO. 167, nucleotide sequence of  Mutant ID 37 (OptiTaq KlenTaq)CATATGGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGGTCCATGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC. SEQ ID NO. 168, amino acid sequence of Mutant ID 37 (OptiTaq KlenTaq)MGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDW LSAKEAA.SEQ ID NO. 169, nucleotide sequence of Mutant ID 38 (A661E, I665W, F667L KlenTaq)CATATGGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCC GT GAA GCTAAAACA TGGAAT TTG GGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGCGCACGTATGCTTCTGCAGGTCCATGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC. SEQ ID NO. 170, amino acid sequence of Mutant ID 38 (A661E, I665W, F667L KlenTaq)MGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRR E AKT W N L GVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDW LSAKEAA.SEQ ID NO. 171, nucleotide sequence of  Mutant ID 39 (V783F KlenTaq)CATATGGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGC GCACGTATGCTTCTGCAGTTC CATGACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC. SEQ ID NO. 172, amino acid sequence of Mutant ID 39 (V783F KlenTaq)MGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVERLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGA RMLLQ LHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDW LSAKEAA.SEQ ID NO. 173, nucleotide sequence of  Mutant ID 40 (H784Q KlenTaq)CATATGGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGC GCACGTATGCTTCTGCAGGTCCAG GACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC. SEQ ID NO. 174, amino acid sequence of Mutant ID 40 (H784Q KlenTaq)MGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGA RMLLQV QDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDW LSAKEAA.SEQ ID NO. 175, nucleotide sequence of Mutant ID 41 (V783L H784Q KlenTaq)CATATGGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGC GCACGTATGCTTCTGCAGCTGCAG GACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC. SEQ ID NO. 176, amino acid sequence of Mutant ID 41 (V783L H784Q KlenTaq)MGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGA RMLLQ LHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDW LSAKEAA.SEQ ID NO. 177, nucleotide sequence of  Mutant ID 42 (H784S KlenTaq)CATATGGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGC GCACGTATGCTTCTGCAGGTCAGC GACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC. SEQ ID NO. 178, amino acid sequence of Mutant ID 42 (H784S KlenTaq)MGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGA RMLLQV SDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDW LSAKEAA.SEQ ID NO. 179, nucleotide sequence of  Mutant ID 43 (H784Y KlenTaq)CATATGGGTTCACTGCTTCATGAATTCGGTCTGTTAGAGTCTCCTAAAGCACTCGAAGAGGCACCGTGGCCGCCCCCAGAAGGTGCTTTTGTTGGCTTCGTACTTTCCCGTAAGGAGCCTATGTGGGCAGATCTTCTGGCTTTAGCGGCTGCACGCGGTGGCCGTGTTCACCGGGCCCCTGAGCCATACAAAGCGTTACGTGATCTGAAGGAAGCACGTGGCTTGCTGGCAAAAGACCTTTCTGTTTTGGCCCTGCGCGAGGGTCTTGGACTGCCGCCAGGCGACGATCCCATGTTATTGGCCTATCTGTTAGACCCTAGCAATACCACACCTGAAGGGGTCGCTCGTCGGTATGGCGGTGAATGGACTGAGGAAGCCGGAGAGCGCGCCGCATTGTCCGAACGGCTCTTTGCAAACTTATGGGGTCGTCTGGAAGGGGAGGAACGTCTGTTATGGTTGTATCGGGAAGTCGAACGTCCTCTTTCGGCCGTATTAGCGCATATGGAGGCAACAGGTGTGCGTTTAGATGTCGCGTACCTTCGGGCCTTATCACTGGAAGTTGCAGAGGAAATCGCCCGTCTCGAGGCTGAAGTGTTCCGGTTGGCCGGTCACCCGTTTAACCTCAACTCCCGTGACCAGCTGGAACGCGTTTTATTCGATGAGCTTGGGCTTCCCGCAATTGGCAAAACCGAAAAGACTGGCAAACGCAGTACGAGCGCTGCCGTCCTTGAGGCACTCCGCGAGGCTCACCCTATTGTAGAAAAGATCCTGCAATACCGTGAGTTGACGAAGCTTAAAAGCACTTATATTGATCCTCTCCCGGATCTGATCCATCCTCGTACCGGCCGCTTGCACACACGTTTCAACCAGACGGCGACTGCAACCGGCCGTCTGTCTAGCTCGGATCCAAATCTCCAGAACATTCCGGTCCGTACACCCTTGGGCCAACGTATCCGCCGGGCGTTTATCGCTGAGGAAGGATGGTTACTGGTCGCATTGGACTACTCGCAGATTGAGCTGCGCGTCCTCGCACATCTCTCTGGTGACGAAAATTTAATCCGCGTGTTTCAAGAGGGGCGTGATATTCACACAGAAACTGCCTCATGGATGTTCGGTGTCCCACGTGAAGCAGTGGATCCTTTGATGCGCCGTGCAGCTAAAACAATTAATTTTGGAGTGCTGTACGGAATGAGCGCTCATCGCTTGAGTCAGGAACTGGCAATCCCCTACGAGGAAGCGCAGGCATTCATCGAACGTTACTTTCAATCGTTTCCGAAAGTTCGCGCATGGATCGAGAAGACGCTCGAGGAAGGTCGTCGTCGGGGCTATGTCGAAACTCTGTTTGGTCGCCGTCGGTACGTACCAGATCTTGAAGCCCGCGTCAAATCGGTACGGGAGGCTGCGGAGCGTATGGCATTTAATATGCCTGTACAGGGTACTGCAGCTGACCTCATGAAACTGGCAATGGTCAAGCTTTTCCCGCGCTTGGAGGAAATGGGC GCACGTATGCTTCTGCAGGTCTAT GACGAGCTGGTGTTAGAAGCCCCTAAGGAGCGCGCCGAAGCTGTCGCGCGCCTCGCTAAAGAAGTGATGGAGGGCGTTTACCCATTGGCCGTACCCCTCGAAGTGGAGGTCGGTATTGGAGAAGATTGGTTATCTGCAAAGGAAGCGGCCGC. SEQ ID NO. 180, amino acid sequence of Mutant ID 43 (H784Y KlenTaq)MGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEETARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGA RMLLQV YDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDW LSAKEAA.

INCORPORATION BY REFERENCE

All publications, patents, patent applications, Accession No. datamentioned herein are hereby incorporated by reference in their entiretyas if each individual publication, patent, patent application orAccession No. data was specifically and individually indicated to beincorporated by reference. In the case of Accession No. data citationsand references, the corresponding DNA polymerase amino acid andnucleotide sequences are incorporated herein by reference as if suchsequences are disclosed by way of a SEQ ID NO. In case of conflict, thepresent application, including any definitions herein, will control.

The terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. With respect tothe use of substantially, any plural and/or singular terms herein, thosehaving skill in the art can translate from the plural as is appropriateto the context and/or application. The various singular/pluralpermutations may be expressly set forth herein for the sake of clarity.

While the present invention has been described with reference to certainembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted withoutdeparting from the scope of the present invention. In addition, manymodifications may be made to adapt a particular situation or material tothe teachings of the present invention without departing from its scope.Therefore, it is intended that the present invention not be limited tothe particular embodiments or examples disclosed, but that the presentinvention will include all embodiments falling within the scope of theappended claims.

What is claimed is:
 1. A mutant Taq DNA polymerase having an enhancedtemplate discrimination activity compared with an unmodified Taq DNApolymerase of SEQ ID NO.:1, wherein the amino acid sequence of themutant Taq DNA polymerase consists of substitutions at residue positions783, 784, or a combination of 783 and 784 of the unmodified Taq DNApolymerase of SEQ ID NO.:1.
 2. The mutant Taq DNA polymerase of claim 1,wherein the enhanced template discrimination activity comprises at leastone property selected from the group consisting of enhanced 3′-mismatchdiscrimination and enhanced 3′-nucleotide discrimination.
 3. The mutantTaq DNA polymerase of claim 1, further comprising polymerase activity atleast comparable to about 0.01-fold the polymerase activity ofunmodified Taq DNA polymerase.
 4. The mutant Taq DNA polymerase of claim1, wherein the enhanced template discrimination activity comprisesenhanced 3′-mismatch discrimination, wherein an average ΔΔCq is at leastabout 1.0 when the mutant Taq DNA polymerase is evaluated for enhanced3′-mismatch discrimination by allele-specific PCR assay.
 5. The mutantTaq DNA polymerase of claim 1, wherein the enhanced templatediscrimination activity comprises enhanced 3′-nucleotide discrimination,wherein an average ΔΔCq is at least about 1.0 when the mutant Taq DNApolymerase is evaluated for enhanced 3′-nucleotide discrimination byquantitative PCR.
 6. The mutant Taq DNA polymerase of claim 1, whereinthe enhanced template discrimination activity comprises enhanced3′-mismatch discrimination by rhPCR, wherein an average ΔΔCq is at leastabout 1.0 when the mutant Taq DNA polymerase is evaluated for enhanced3′-mismatch discrimination by rhPCR with an RDDDDx blocked-cleavablerhPCR primer.
 7. The mutant Taq DNA polymerase of claim 1, wherein theenhanced template discrimination activity comprises enhanced 3′-mismatchdiscrimination by rhPCR, wherein an average ΔΔCq is at least about 0.50when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatchdiscrimination by rhPCR with an RDxxD blocked-cleavable rhPCR primer. 8.The mutant Taq DNA polymerase of claim 1, wherein the enhanced templatediscrimination activity comprises enhanced 3′-mismatch discrimination byrhPCR SNP discrimination assay, wherein an average ΔΔCq is at leastabout 1.0 when the mutant Taq DNA polymerase is evaluated for enhanced3′-mismatch discrimination by rhPCR SNP discrimination assay of theSMAD7 gene NM_005904, C/T SNP, rs4939827) with RDDDDx blocked-cleavablerhPCR primers consisting of SEQ ID NOs:76 and
 77. 9. The mutant Taq DNApolymerase of claim 1, wherein the enhanced template discriminationactivity comprises enhanced 3′-mismatch discrimination by rhPCR SNPdiscrimination assay, wherein an average ΔΔCq is at least about 1.0 whenthe mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatchdiscrimination by rhPCR SNP discrimination assay of the SMAD7 geneNM_005904, C/T SNP, rs4939827) with RDxxD blocked-cleavable rhPCRprimers consisting of SEQ ID NOs:78 and
 79. 10. The mutant Taq DNApolymerase of claim 1, wherein the enhanced template discriminationactivity comprises enhanced 3′-mismatch discrimination by rhPCR SNPdiscrimination assay, wherein an average ΔΔCq is at least about 5.0 whenthe mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatchdiscrimination by rhPCR SNP discrimination assay of the SMAD7 geneNM_005904, C/T SNP, rs4939827) with RDxxD blocked-cleavable rhPCRprimers consisting of SEQ ID NOs:78 and
 79. 11. The mutant Taq DNApolymerase of claim 1, wherein the enhanced template discriminationactivity comprises enhanced rare allele discrimination, wherein theenhanced rare allele discrimination is at least 1:10,000 when the mutantTaq DNA polymerase is evaluated by rhPCR SNP discrimination assay of theSMAD7 gene NM_005904, C/T SNP, rs4939827) having a ΔCq of at least 3.0with RDxxD blocked-cleavable rhPCR primers consisting of SEQ ID NOs:78and
 79. 12. A mutant Taq DNA polymerase having an enhanced templatediscrimination activity compared with an unmodified Taq DNA polymerase,wherein the amino acid sequence of the mutant Taq DNA polymerase isselected from the group of the following selected substitutions: V783F,H784Q, or a combination of V783L and H784Q.
 13. The mutant Taq DNApolymerase of claim 12, wherein the mutant Taq DNA polymerase has atleast 80% sequence identity to one of SEQ ID NOS: 83, 85, 87 or
 89. 14.The mutant Taq DNA polymerase of claim 12, wherein the enhanced templatediscrimination activity comprises at least one property selected fromthe group consisting of enhanced 3′-mismatch discrimination and enhanced3′-nucleotide discrimination.
 15. The mutant Taq DNA polymerase of claim12, further comprising polymerase activity at least comparable to about0.02-fold the polymerase activity of unmodified Taq DNA polymerase. 16.The mutant Taq DNA polymerase of claim 12, wherein the enhanced templatediscrimination activity comprises enhanced 3′-mismatch discrimination,wherein an average ΔΔCq is at least about 5.0 when the mutant Taq DNApolymerase is evaluated for enhanced 3′-mismatch discrimination byallele-specific PCR assay.
 17. The mutant Taq DNA polymerase of claim12, wherein the enhanced template discrimination activity comprisesenhanced 3′-nucleotide discrimination, wherein an average ΔΔCq is atleast about 3.0 when the mutant Taq DNA polymerase is evaluated forenhanced 3′-nucleotide discrimination by quantitative PCR.
 18. Themutant Taq DNA polymerase of claim 12, wherein the enhanced templatediscrimination activity comprises enhanced 3′-mismatch discrimination,wherein an average ΔΔCq is at least about 1.0 when the mutant Taq DNApolymerase is evaluated for enhanced 3′-mismatch discrimination by rhPCRwith an RDDDDx blocked-cleavable rhPCR primer.
 19. The mutant Taq DNApolymerase of claim 12, wherein the enhanced template discriminationactivity comprises enhanced 3′-mismatch discrimination, wherein anaverage ΔΔCq is at least about 0.60 when the mutant Taq DNA polymeraseis evaluated for enhanced 3′-mismatch discrimination by rhPCR with anRDxxD blocked-cleavable rhPCR primer.
 20. The mutant Taq DNA polymeraseof claim 12, wherein the enhanced template discrimination activitycomprises enhanced 3′-mismatch discrimination by rhPCR SNPdiscrimination assay, wherein an average ΔΔCq is at least about 1.0 whenthe mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatchdiscrimination by rhPCR SNP discrimination assay of the SMAD7 geneNM_005904, C/T SNP, rs4939827) with RDDDDx blocked-cleavable rhPCRprimers consisting of SEQ ID NOs:76 and
 77. 21. The mutant Taq DNApolymerase of claim 12, wherein the enhanced template discriminationactivity comprises enhanced 3′-mismatch discrimination by rhPCR SNPdiscrimination assay, wherein an ΔΔCq is at least about 3.5 when themutant Taq DNA polymerase is evaluated for enhanced 3′-mismatchdiscrimination by rhPCR SNP discrimination assay of the SMAD7 geneNM_005904, C/T SNP, rs4939827) with RDxxD blocked-cleavable rhPCRprimers consisting of SEQ ID NOs:78 and
 79. 22. The mutant Taq DNApolymerase of claim 12, wherein the enhanced template discriminationactivity comprises enhanced 3′-mismatch discrimination by rhPCR SNPdiscrimination assay, wherein an ΔΔCq is at least about 15.0 when themutant Taq DNA polymerase is evaluated for enhanced 3′-mismatchdiscrimination by rhPCR SNP discrimination assay of the SMAD7 geneNM_005904, C/T SNP, rs4939827) with RDxxD blocked-cleavable rhPCRprimers consisting of SEQ ID NOs:78 and
 79. 23. The mutant Taq DNApolymerase of claim 12, wherein the enhanced template discriminationactivity comprises enhanced rare allele discrimination, wherein theenhanced rare allele discrimination is at least 1:10,000 when the mutantTaq DNA polymerase is evaluated by rhPCR SNP discrimination assay of theSMAD7 gene NM_005904, C/T SNP, rs4939827) having a ΔCq of at least 3.0with RDxxD blocked-cleavable rhPCR primers consisting of SEQ ID NOs:78and
 79. 24. A kit for producing an extended primer, comprising: at leastone container providing a mutant DNA polymerase according to claim 1.25. The kit according to claim 24, further comprising one or moreadditional containers selected from the group consisting of: (a) acontainer providing a primer hybridizable, under primer extensionconditions, to a predetermined polynucleotide template; (b) a containerproviding nucleoside triphosphates; and (c) a container providing abuffer suitable for primer extension.
 26. The kit according to claim 24,further comprising one or more additional containers selected from thegroup consisting of (a) a container containing a blocked-cleavableprimer and (b) a container containing RNase H2.
 27. A reaction mixturecomprising a mutant DNA polymerase according to claim 1, at least oneprimer, a polynucleotide template, and nucleoside triphosphates.
 28. Thereaction mixture according to claim 27, wherein the at least one primercomprises a blocked-cleavable primer.
 29. The reaction mixture accordingto claim 27, further comprising RNase H2.
 30. A method for performingrhPCR, comprising performing primer extension with a mutant DNApolymerase of claim
 1. 31. A method for performing rhPCR, comprisingperforming primer extension with a mutant Taq DNA polymerase of claim12.
 32. A mutant Taq DNA polymerase consisting of an amino acid sequenceselected from a group consisting of SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:89, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:155, SEQ ID NO:151, SEQID NO:153, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO: 172,SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, and SEQ ID NO: 180.